A versatile assay for RNA-binding proteins in living cells.
Strein, C., Alleaume, A.M., Rothbauer, U., Hentze, M.W. & Castello, A.
RNA. 2014 Mar 24.
RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.
mRNA 3'end processing: A tale of the tail reaches the clinic.
Hollerer, I., Grund, K., Hentze, M.W. & Kulozik, A.E.
EMBO Mol Med. 2014 Jan 1;6(1):16-26. doi: 10.1002/emmm.201303300.
Recent advances reveal mRNA 3'end processing as a highly regulated process that fine-tunes posttranscriptional gene expression. This process can affect the site and/or the efficiency of 3'end processing, controlling the quality and the quantity of substrate mRNAs. The regulation of 3'end processing plays a central role in fundamental physiology such as blood coagulation and innate immunity. In addition, errors in mRNA 3'end processing have been associated with a broad spectrum of human diseases, including cancer. We summarize and discuss the paradigmatic shift in the understanding of 3'end processing as a mechanism of posttranscriptional gene regulation that has reached clinical medicine.
Unbiased RNAi screen for hepcidin regulators links hepcidin suppression to proliferative Ras/RAF and nutrient-dependent mTOR signaling.
Mleczko-Sanecka, K., Roche, F., da Silva, A.R., Call, D., D'Alessio, F., Ragab, A., Lapinski, P.E., Ummanni, R., Korf, U., Oakes, C., Damm, G., D'Alessandro, L.A., Klingmuller, U., King, P.D., Boutros, M., Hentze, M.W. & Muckenthaler, M.U.
Blood. 2014 Mar 6;123(10):1574-85. doi: 10.1182/blood-2013-07-515957. Epub 2014Jan 2.
The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.
Iron regulatory protein-1 protects against mitoferrin-1-deficient porphyria.
Chung, J., Anderson, S.A., Gwynn, B., Deck, K.M., Chen, M.J., Langer, N.B., Shaw, G.C., Huston, N.C., Boyer, L.F., Datta, S., Paradkar, P.N., Li, L., Wei, Z., Lambert, A.J., Sahr, K., Wittig, J.G., Chen, W., Lu, W., Galy, B., Schlaeger, T.M., Hentze, M.W., Ward, D.M., Kaplan, J., Eisenstein, R.S., Peters, L.L. & Paw, B.H.
J Biol Chem. 2014 Mar 14;289(11):7835-43. doi: 10.1074/jbc.M114.547778. Epub 2014Feb 7.
Mitochondrial iron is essential for the biosynthesis of heme and iron-sulfur ([Fe-S]) clusters in mammalian cells. In developing erythrocytes, iron is imported into the mitochondria by MFRN1 (mitoferrin-1, SLC25A37). Although loss of MFRN1 in zebrafish and mice leads to profound anemia, mutant animals showed no overt signs of porphyria, suggesting that mitochondrial iron deficiency does not result in an accumulation of protoporphyrins. Here, we developed a gene trap model to provide in vitro and in vivo evidence that iron regulatory protein-1 (IRP1) inhibits protoporphyrin accumulation. Mfrn1(+/gt);Irp1(-/-) erythroid cells exhibit a significant increase in protoporphyrin levels. IRP1 attenuates protoporphyrin biosynthesis by binding to the 5'-iron response element (IRE) of alas2 mRNA, inhibiting its translation. Ectopic expression of alas2 harboring a mutant IRE, preventing IRP1 binding, in Mfrn1(gt/gt) cells mimics Irp1 deficiency. Together, our data support a model whereby impaired mitochondrial [Fe-S] cluster biogenesis in Mfrn1(gt/gt) cells results in elevated IRP1 RNA-binding that attenuates ALAS2 mRNA translation and protoporphyrin accumulation.
Pathologies at the nexus of blood coagulation and inflammation: thrombin in hemostasis, cancer, and beyond.
Danckwardt, S., Hentze, M.W. & Kulozik, A.E.
J Mol Med (Berl). 2013 Nov;91(11):1257-71. doi: 10.1007/s00109-013-1074-5. Epub2013 Aug 17.
Thrombin is the protease involved in blood coagulation. Its deregulation can lead to hemostatic abnormalities, which range from subtle subclinical to serious life-threatening coagulopathies, i.e., during septicemia. Additionally, thrombin plays important roles in many (patho)physiological conditions that reach far beyond its well-established role in stemming blood loss and thrombosis, including embryonic development and angiogenesis but also extending to inflammatory processes, complement activation, and even tumor biology. In this review, we will address thrombin's broad roles in diverse (patho)physiological processes in an integrative way. We will also discuss thrombin as an emerging major target for novel therapies.
The RNA-binding protein repertoire of embryonic stem cells.
Kwon, S.C., Yi, H., Eichelbaum, K., Fohr, S., Fischer, B., You, K.T., Castello, A., Krijgsveld, J., Hentze, M.W. & Kim, V.N.
Nat Struct Mol Biol. 2013 Sep;20(9):1122-30. doi: 10.1038/nsmb.2638. Epub 2013Aug 4.
RNA-binding proteins (RBPs) have essential roles in RNA-mediated gene regulation, and yet annotation of RBPs is limited mainly to those with known RNA-binding domains. To systematically identify the RBPs of embryonic stem cells (ESCs), we here employ interactome capture, which combines UV cross-linking of RBP to RNA in living cells, oligo(dT) capture and MS. From mouse ESCs (mESCs), we have defined 555 proteins constituting the mESC mRNA interactome, including 283 proteins not previously annotated as RBPs. Of these, 68 new RBP candidates are highly expressed in ESCs compared to differentiated cells, implicating a role in stem-cell physiology. Two well-known E3 ubiquitin ligases, Trim25 (also called Efp) and Trim71 (also called Lin41), are validated as RBPs, revealing a potential link between RNA biology and protein-modification pathways. Our study confirms and expands the atlas of RBPs, providing a useful resource for the study of the RNA-RBP network in stem cells.
Abnormal body iron distribution and erythropoiesis in a novel mouse model with inducible gain of iron regulatory protein (IRP)-1 function.
Casarrubea, D., Viatte, L., Hallas, T., Vasanthakumar, A., Eisenstein, R.S., Schumann, K., Hentze, M.W. & Galy, B.
J Mol Med (Berl). 2013 Jul;91(7):871-81. doi: 10.1007/s00109-013-1008-2. Epub2013 Mar 1.
Disorders of iron metabolism account for some of the most common human diseases. Cellular iron homeostasis is maintained by iron regulatory proteins (IRP)-1 and 2 through their binding to cis-regulatory iron-responsive elements (IREs) in target mRNAs. Mouse models with IRP deficiency have yielded valuable insights into iron biology, but the physiological consequences of gain of IRP function in mammalian organisms have remained unexplored. Here, we report the generation of a mouse line allowing conditional expression of a constitutively active IRP1 mutant (IRP1) using Cre/Lox technology. Systemic activation of the IRP1 transgene from the Rosa26 locus yields viable animals with gain of IRE-binding activity in all the organs analyzed. IRP1 activation alters the expression of IRP target genes and is accompanied by iron loading in the same organs. Furthermore, mice display macrocytic erythropenia with decreased hematocrit and hemoglobin levels as well as impaired erythroid differentiation. Thus, inappropriately high IRP1 activity causes disturbed body iron distribution and erythropoiesis. This new mouse model further highlights the importance of appropriate IRP regulation in central organs of iron metabolism. Moreover, it opens novel avenues to study diseases associated with abnormally high IRP1 activity, such as Parkinson's disease or Friedreich's ataxia.
Making sense of nonsense.
Hentze, M.W. & Izaurralde, E.
Nat Struct Mol Biol. 2013 Jun;20(6):651-3. doi: 10.1038/nsmb.2601. Europe PMC
RNA-binding proteins in Mendelian disease.
Castello, A., Fischer, B., Hentze, M.W. & Preiss, T.
Trends Genet. 2013 May;29(5):318-27. doi: 10.1016/j.tig.2013.01.004. Epub 2013Feb 15.
RNA-binding proteins (RBPs) control all aspects of RNA fate, and defects in their function underlie a broad spectrum of human pathologies. We focus here on two recent studies that uncovered the in vivo mRNA interactomes of human cells, jointly implicating over 1100 proteins in RNA binding. Surprisingly, over 350 of these RBPs had no prior RNA binding-related annotation or domain homology. The datasets also contain many proteins that, when mutated, cause Mendelian diseases, prominently neurological, sensory, and muscular disorders and cancers. Disease mutations in these proteins occur throughout their domain architectures and many are found in non-classical RNA-binding domains and in disordered regions. In some cases, mutations might cause disease through perturbing previously unknown RNA-related protein functions. These studies have thus expanded our knowledge of RBPs and their role in genetic diseases. We also expect that mRNA interactome capture approaches will aid further exploration of RNA systems biology in varied physiological and pathophysiological settings.
Circular RNAs: splicing's enigma variations.
Hentze, M.W. & Preiss, T.
EMBO J. 2013 Apr 3;32(7):923-5. doi: 10.1038/emboj.2013.53. Epub 2013 Mar 5. Europe PMC
Iron regulatory proteins control a mucosal block to intestinal iron absorption.
Galy, B., Ferring-Appel, D., Becker, C., Gretz, N., Grone, H.J., Schumann, K. & Hentze, M.W.
Cell Rep. 2013 Mar 28;3(3):844-57. doi: 10.1016/j.celrep.2013.02.026. Epub 2013Mar 21.
Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated "mucosal block." We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin "mucosal block" and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.
The IRP1-HIF-2alpha axis coordinates iron and oxygen sensing with erythropoiesis and iron absorption.
Anderson, S.A., Nizzi, C.P., Chang, Y.I., Deck, K.M., Schmidt, P.J., Galy, B., Damnernsawad, A., Broman, A.T., Kendziorski, C., Hentze, M.W., Fleming, M.D., Zhang, J. & Eisenstein, R.S.
Cell Metab. 2013 Feb 5;17(2):282-90. doi: 10.1016/j.cmet.2013.01.007.
Red blood cell production is a finely tuned process that requires coordinated oxygen- and iron-dependent regulation of cell differentiation and iron metabolism. Here, we show that translational regulation of hypoxia-inducible factor 2alpha (HIF-2alpha) synthesis by iron regulatory protein 1 (IRP1) is critical for controlling erythrocyte number. IRP1-null (Irp1(-/-)) mice display a marked transient polycythemia. HIF-2alpha messenger RNA (mRNA) is derepressed in kidneys of Irp1(-/-) mice but not in kidneys of Irp2(-/-) mice, leading to increased renal erythropoietin (Epo) mRNA and inappropriately elevated serum Epo levels. Expression of the iron transport genes DCytb, Dmt1, and ferroportin, as well as other HIF-2alpha targets, is enhanced in Irp1(-/-) duodenum. Analysis of mRNA translation state in the liver revealed IRP1-dependent dysregulation of HIF-2alpha mRNA translation, whereas IRP2 deficiency derepressed translation of all other known 5' iron response element (IRE)-containing mRNAs expressed in the liver. These results uncover separable physiological roles of each IRP and identify IRP1 as a therapeutic target for manipulating HIF-2alpha action in hematologic, oncologic, and other disorders.
System-wide identification of RNA-binding proteins by interactome capture.
Castello, A., Horos, R., Strein, C., Fischer, B., Eichelbaum, K., Steinmetz, L.M., Krijgsveld, J. & Hentze, M.W.
Nat Protoc. 2013 Feb 14;8(3):491-500. doi: 10.1038/nprot.2013.020. Epub 2013 Feb14.
Owing to their preeminent biological functions, the repertoire of expressed RNA-binding proteins (RBPs) and their activity states are highly informative about cellular systems. We have developed a novel and unbiased technique, called interactome capture, for identifying the active RBPs of cultured cells. By making use of in vivo UV cross-linking of RBPs to polyadenylated RNAs, covalently bound proteins are captured with oligo(dT) magnetic beads. After stringent washes, the mRNA interactome is determined by quantitative mass spectrometry (MS). The protocol takes 3 working days for analysis of single proteins by western blotting, and about 2 weeks for the determination of complete cellular mRNA interactomes by MS. The most important advantage of interactome capture over other in vitro and in silico approaches is that only RBPs bound to RNA in a physiological environment are identified. When applied to HeLa cells, interactome capture revealed hundreds of novel RBPs. Interactome capture can also be broadly used to compare different biological states, including metabolic stress, cell cycle, differentiation, development or the response to drugs.
The hemochromatosis proteins HFE, TfR2, and HJV form a membrane-associated protein complex for hepcidin regulation.
D'Alessio, F., Hentze, M.W. & Muckenthaler, M.U.
J Hepatol. 2012 Nov;57(5):1052-60. doi: 10.1016/j.jhep.2012.06.015. Epub 2012 Jun21.
BACKGROUND & AIMS: The hereditary hemochromatosis-associated membrane proteins HFE, TfR2, and HJV are required for adequate hepatic expression of the iron hormone hepcidin. While the genetic interactions are clear, it remains elusive how bone morphogenetic protein co-receptor HJV functions together with HFE and TfR2 to activate hepcidin transcription via the BMP-SMAD signaling pathway. Here, we investigate whether HFE, TfR2, and HJV physically interact on the surface of hepatocytes. METHODS: We explore protein-protein interactions by glycerol gradient sedimentation assays and co-immunoprecipitation analyses in transfected HuH7 hepatoma-derived cells. RESULTS: Our data demonstrate that HFE and TfR2 bind HJV in a non-competitive manner. Co-immunoprecipitation analyses provide direct experimental evidence that HFE, TfR2, and HJV form a multi-protein membrane complex. Our experiments show that like TfR2, HJV competes with TfR1 for binding to HFE, indicating that the expression of TfR2 and HJV may be critical for iron sensing. We identify residues 120-139 of the TfR2 extra-cellular domain as the critical amino acids required for the binding of both HFE and HJV. Interestingly, RGMA, a central nervous system homolog, can substitute for HJV in the complex and promote hepcidin transcription, implicating RGMA in the local control of hepcidin in the CNS. CONCLUSIONS: Taken together, our findings provide a biochemical basis for hepcidin control by HFE, TfR2, and HJV.
From cis-regulatory elements to complex RNPs and back.
Gebauer, F., Preiss, T. & Hentze, M.W.
Cold Spring Harb Perspect Biol. 2012 Jul 1;4(7):a012245. doi:10.1101/cshperspect.a012245.
Messenger RNAs (mRNAs), the templates for translation, have evolved to harbor abundant cis-acting sequences that affect their posttranscriptional fates. These elements are frequently located in the untranslated regions and serve as binding sites for trans-acting factors, RNA-binding proteins, and/or small non-coding RNAs. This article provides a systematic synopsis of cis-acting elements, trans-acting factors, and the mechanisms by which they affect translation. It also highlights recent technical advances that have ushered in the era of transcriptome-wide studies of the ribonucleoprotein complexes formed by mRNAs and their trans-acting factors.
PABP and the poly(A) tail augment microRNA repression by facilitated miRISC binding.
Moretti, F., Kaiser, C., Zdanowicz-Specht, A. & Hentze, M.W.
Nat Struct Mol Biol. 2012 May 27;19(6):603-8. doi: 10.1038/nsmb.2309.
Polyadenylated mRNAs are typically more strongly repressed by microRNAs (miRNAs) than their nonadenylated counterparts. Using a Drosophila melanogaster cell-free translation system, we found that this effect is mediated by the poly(A)-binding protein (PABP). miRNA repression was positively correlated with poly(A) tail length, but this stimulatory effect on repression was lost when translation was repressed by the tethered GW182 silencing domain rather than the miRNA-induced silencing complex (miRISC) itself. These findings are mechanistically explained by a notable function of PABP: it promotes association of miRISC with miRNA-regulated mRNAs. We also found that PABP association with mRNA rapidly diminished with miRISC recruitment and before detectable deadenylation. We integrated these data into a revised model for the function of PABP and the poly(A) tail in miRNA-mediated translational repression.
Insights into RNA Biology from an Atlas of Mammalian mRNA-Binding Proteins.
Castello, A., Fischer, B., Eichelbaum, K., Horos, R., Beckmann, B.M., Strein, C., Davey, N.E., Humphreys, D.T., Preiss, T., Steinmetz, L.M., Krijgsveld, J. & Hentze, M.W.
Cell. 2012 Jun 8;149(6):1393-406. Epub 2012 May 31.
RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings.
Automated High-Throughput RNAi Screening in Human Cells Combined with Reporter mRNA Transfection to Identify Novel Regulators of Translation.
Casanova, C.M., Sehr, P., Putzker, K., Hentze, M.W., Neumann, B., Duncan, K.E. & Thoma, C.
PLoS One. 2012;7(9):e45943. doi: 10.1371/journal.pone.0045943. Epub 2012 Sep 27.
Proteins that promote angiogenesis, such as vascular endothelial growth factor (VEGF), are major targets for cancer therapy. Accordingly, proteins that specifically activate expression of factors like VEGF are potential alternative therapeutic targets and may help to combat evasive resistance to angiogenesis inhibitors. VEGF mRNA contains two internal ribosome entry sites (IRESs) that enable selective activation of VEGF protein synthesis under hypoxic conditions that trigger angiogenesis. To identify novel regulators of VEGF IRES-driven translation in human cells, we have developed a high-throughput screening approach that combines siRNA treatment with transfection of a VEGF-IRES reporter mRNA. We identified the kinase MAPK3 as a novel positive regulator of VEGF IRES-driven translation and have validated its regulatory effect on endogenous VEGF. Our automated method is scalable and readily adapted for use with other mRNA regulatory elements. Consequently, it should be a generally useful approach for high-throughput identification of novel regulators of mRNA translation.
Iron regulatory protein-1 and -2: transcriptome-wide definition of binding mRNAs and shaping of the cellular proteome by iron regulatory proteins.
Sanchez, M., Galy, B., Schwanhaeusser, B., Blake, J., Bahr-Ivacevic, T., Benes, V., Selbach, M., Muckenthaler, M.U. & Hentze, M.W.
Blood. 2011 Nov 24;118(22):e168-79. Epub 2011 Sep 22.
Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.
Translational Control via Protein-Regulated Upstream Open Reading Frames.
Medenbach, J., Seiler, M. & Hentze, M.W.
Cell. 2011 Jun 10;145(6):902-13.
Analysis of the regulation of msl-2 mRNA by Sex lethal (SXL), which is critical for dosage compensation in Drosophila, has uncovered a mode of translational control based on common 5' untranslated region elements, upstream open reading frames (uORFs), and interaction sites for RNA-binding proteins. We show that SXL binding downstream of a short uORF imposes a strong negative effect on major reading frame translation. The underlying mechanism involves increasing initiation of scanning ribosomes at the uORF and augmenting its impediment to downstream translation. Our analyses reveal that SXL exerts its effect controlling initiation, not elongation or termination, at the uORF. Probing the generality of the underlying mechanism, we show that the regulatory module that we define experimentally functions in a heterologous context, and we identify natural Drosophila mRNAs that are regulated via this module. We propose that protein-regulated uORFs constitute a systematic principle for the regulation of protein synthesis.
An efficient factor-depleted mammalian in vitro translation system.
Rakotondrafara, A.M. & Hentze, M.W.
Nat Protoc. 2011 May;6(5):563-71. Epub 2011 Apr 7.
Much of the regulation of gene expression occurs at the level of protein synthesis. In addition to the canonical translation factors, a multitude of proteins and microRNAs (miRNAs) act as regulatory trans-acting factors. Mechanistic analysis of translational control benefits from functional cell-free systems that can be depleted of the responsible regulatory factors. Although antisense oligonucleotides facilitate the functional sequestration of the regulatory RNAs, immunodepletion of protein factors is technically challenging. Here we describe a simple and robust alternative protocol for the preparation of factor-depleted in vitro translation system derived from HeLa cells. The procedure relies on RNA interference-mediated knockdown of the factor of interest prior to extract preparation, and it overcomes problems with the availability and specificity of antibodies, as well as with the co-depletion of proteins associated with the factor under study. The complete procedure can normally be conducted within 1 week and carried out in parallel for multiple (candidate) factors.
The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice.
Castoldi, M., Vujic Spasic, M., Altamura, S., Elmen, J., Lindow, M., Kiss, J., Stolte, J., Sparla, R., D'Alessandro, L.A., Klingmuller, U., Fleming, R.E., Longerich, T., Grone, H.J., Benes, V., Kauppinen, S., Hentze, M.W. & Muckenthaler, M.U.
J Clin Invest. 2011 Apr;121(4):1386-96. doi: 10.1172/JCI44883.
Systemic iron homeostasis is mainly controlled by the liver through synthesis of the peptide hormone hepcidin (encoded by Hamp), the key regulator of duodenal iron absorption and macrophage iron release. Here we show that the liver-specific microRNA miR-122 is important for regulating Hamp mRNA expression and tissue iron levels. Efficient and specific depletion of miR-122 by injection of a locked-nucleic-acid-modified (LNA-modified) anti-miR into WT mice caused systemic iron deficiency, characterized by reduced plasma and liver iron levels, mildly impaired hematopoiesis, and increased extramedullary erythropoiesis in the spleen. Moreover, miR-122 inhibition increased the amount of mRNA transcribed by genes that control systemic iron levels, such as hemochromatosis (Hfe), hemojuvelin (Hjv), bone morphogenetic protein receptor type 1A (Bmpr1a), and Hamp. Importantly, miR-122 directly targeted the 3' untranslated region of 2 mRNAs that encode activators of hepcidin expression, Hfe and Hjv. These data help to explain the increased Hamp mRNA levels and subsequent iron deficiency in mice with reduced miR-122 levels and establish a direct mechanistic link between miR-122 and the regulation of systemic iron metabolism.
p38 MAPK controls prothrombin expression by regulated RNA 3' end processing.
Danckwardt, S., Gantzert, A.S., Macher-Goeppinger, S., Probst, H.C., Gentzel, M., Wilm, M., Grone, H.J., Schirmacher, P., Hentze, M.W. & Kulozik, A.E.
Mol Cell. 2011 Feb 4;41(3):298-310.
Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscriptional regulatory mechanism responding to stress and inflammation. This mechanism is triggered by external stimuli that activate p38 MAPK. In turn, p38 MAPK upmodulates canonical 3' end processing components and phosphorylates the RNA-binding proteins FBP2 and FBP3, which inhibit 3' end processing of mRNAs, such as prothrombin mRNA, that bear a defined upstream sequence element (USE) in their 3'UTRs. Upon phosphorylation, FBP2 and FBP3 dissociate from the USE, making it accessible to proteins that stimulate 3' end processing. We provide in vivo evidence suggesting the importance of this mechanism in inflammatory hypercoagulation and tumor invasion. Regulated 3' end processing thus emerges as a key mechanism of gene regulation with broad biological and medical implications.
Mechanism of translational regulation by miR-2 from sites in the 5' untranslated region or the open reading frame.
Moretti, F., Thermann, R. & Hentze, M.W.
RNA. 2010 Dec;16(12):2493-502. Epub 2010 Oct 21.
MicroRNAs (miRs) commonly regulate translation from target mRNA 3' untranslated regions (UTRs). While effective miR-binding sites have also been identified in 5' untranslated regions (UTRs) or open reading frames (ORFs), the mechanism(s) of miR-mediated regulation from these sites has not been defined. Here, we systematically investigate how the position of miR-binding sites influences translational regulation and characterize their mechanistic basis. We show that specific translational regulation is elicited in vitro and in vivo not only from the 3'UTR, but equally effectively from six Drosophila miR-2-binding sites in the 5'UTR or the ORF. In all cases, miR-2 triggers mRNA deadenylation and inhibits translation initiation in a cap-dependent fashion. In contrast, single or dual miR-2-binding sites in the 5'UTR or the ORF yield rather inefficient or no regulation. This work represents the first demonstration that 5'UTR and ORF miR-binding sites can function mechanistically similarly to the intensively investigated 3'UTR sites. Using single or dual binding sites, it also reveals a biological rationale for the high prevalence of miR regulatory sites in the 3'UTR.
Serum ferritin is derived primarily from macrophages through a nonclassical secretory pathway.
Cohen, L.A., Gutierrez, L., Weiss, A., Leichtmann-Bardoogo, Y., Zhang, D.L., Crooks, D.R., Sougrat, R., Morgenstern, A., Galy, B., Hentze, M.W., Lazaro, F.J., Rouault, T.A. & Meyron-Holtz, E.G.
Blood. 2010 Sep 2;116(9):1574-84. doi: 10.1182/blood-2009-11-253815. Epub 2010May 14.
The serum ferritin concentration is a clinical parameter measured widely for the differential diagnosis of anemia. Its levels increase with elevations of tissue iron stores and with inflammation, but studies on cellular sources of serum ferritin as well as its subunit composition, degree of iron loading and glycosylation have given rise to conflicting results. To gain further understanding of serum ferritin, we have used traditional and modern methodologies to characterize mouse serum ferritin. We find that both splenic macrophages and proximal tubule cells of the kidney are possible cellular sources for serum ferritin and that serum ferritin is secreted by cells rather than being the product of a cytosolic leak from damaged cells. Mouse serum ferritin is composed mostly of L-subunits, whereas it contains few H-subunits and iron content is low. L-subunits of serum ferritin are frequently truncated at the C-terminus, giving rise to a characteristic 17-kD band that has been previously observed in lysosomal ferritin. Taken together with the fact that mouse serum ferritin is not detectably glycosylated, we propose that mouse serum ferritin is secreted through the nonclassical lysosomal secretory pathway.
Iron regulatory proteins secure mitochondrial iron sufficiency and function.
Galy, B., Ferring-Appel, D., Sauer, S.W., Kaden, S., Lyoumi, S., Puy, H., Kolker, S., Grone, H.J. & Hentze, M.W.
Cell Metab. 2010 Aug 4;12(2):194-201.
Mitochondria supply cells with ATP, heme, and iron sulfur clusters (ISC), and mitochondrial energy metabolism involves both heme- and ISC-dependent enzymes. Here, we show that mitochondrial iron supply and function require iron regulatory proteins (IRP), cytosolic RNA-binding proteins that control mRNA translation and stability. Mice lacking both IRP1 and IRP2 in their hepatocytes suffer from mitochondrial iron deficiency and dysfunction associated with alterations of the ISC and heme biosynthetic pathways, leading to liver failure and death. These results uncover a major role of the IRPs in cell biology: to ensure adequate iron supply to the mitochondrion for proper function of this critical organelle.
The REM phase of gene regulation.
Hentze, M.W. & Preiss, T.
Trends Biochem Sci. 2010 Aug;35(8):423-6. Epub 2010 Jun 16.
'Classic' enzymes carry out the housekeeping functions of intermediary metabolism. The past decades have seen a steady trickle of reports of several of these enzymes 'moonlighting' as RNA-binding proteins. Although evidence for a physiological role for RNA binding is strong in a few individual examples, no systematic concept has been proposed for the overall phenomenon. We suggest that these diverse observations might herald the existence of currently hidden post-transcriptional regulatory networks between intermediary metabolism and gene expression based on RNA, enzyme and metabolite interactions. We briefly summarize the evidence in support of such networks and discuss how current approaches can be employed for systematic analyses and integration into our understanding of cellular biology, given the technical and conceptual advances of the 'omics' age.
Two to tango: regulation of Mammalian iron metabolism.
Hentze, M.W., Muckenthaler, M.U., Galy, B. & Camaschella, C.
Cell. 2010 Jul 9;142(1):24-38.
Disruptions in iron homeostasis from both iron deficiency and overload account for some of the most common human diseases. Iron metabolism is balanced by two regulatory systems, one that functions systemically and relies on the hormone hepcidin and the iron exporter ferroportin, and another that predominantly controls cellular iron metabolism through iron-regulatory proteins that bind iron-responsive elements in regulated messenger RNAs. We describe how the two distinct systems function and how they "tango" together in a coordinated manner. We also highlight some of the current questions in mammalian iron metabolism and discuss therapeutic opportunities arising from a better understanding of the underlying biological principles.
SMAD7 controls iron metabolism as a potent inhibitor of hepcidin expression.
Mleczko-Sanecka, K., Casanovas, G., Ragab, A., Breitkopf, K., Muller, A., Boutros, M., Dooley, S., Hentze, M.W. & Muckenthaler, M.U.
Blood. 2010 Apr 1;115(13):2657-65. Epub 2009 Dec 29.
Hepcidin is the master regulatory hormone of systemic iron metabolism. Hepcidin deficiency causes common iron overload syndromes whereas its overexpression is responsible for microcytic anemias. Hepcidin transcription is activated by the bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways, whereas comparatively little is known about how hepcidin expression is inhibited. By using high-throughput siRNA screening we identified SMAD7 as a potent hepcidin suppressor. SMAD7 is an inhibitory SMAD protein that mediates a negative feedback loop to both transforming growth factor-beta and BMP signaling and that recently was shown to be coregulated with hepcidin via SMAD4 in response to altered iron availability in vivo. We show that SMAD7 is coregulated with hepcidin by BMPs in primary murine hepatocytes and that SMAD7 overexpression completely abolishes hepcidin activation by BMPs and transforming growth factor-beta. We identify a distinct SMAD regulatory motif (GTCAAGAC) within the hepcidin promoter involved in SMAD7-dependent hepcidin suppression, demonstrating that SMAD7 does not simply antagonize the previously reported hemojuvelin/BMP-responsive elements. This work identifies a potent inhibitory factor for hepcidin expression and uncovers a negative feedback pathway for hepcidin regulation, providing insight into a mechanism how hepcidin expression may be limited to avoid iron deficiency.
The role of ABCE1 in eukaryotic posttermination ribosomal recycling.
Pisarev, A.V., Skabkin, M.A., Pisareva, V.P., Skabkina, O.V., Rakotondrafara, A.M., Hentze, M.W., Hellen, C.U. & Pestova, T.V.
Mol Cell. 2010 Jan 29;37(2):196-210. doi: 10.1016/j.molcel.2009.12.034.
After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA, and release factor eRF1 and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic posttermination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1, and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg(2+) concentrations. Here, we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg(2+) concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP, and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.
The SXL-UNR corepressor complex uses a PABP-mediated mechanism to inhibit ribosome recruitment to msl-2 mRNA.
Duncan, K.E., Strein, C. & Hentze, M.W.
Mol Cell. 2009 Nov 25;36(4):571-82.
Drosophila female viability requires translational repression of msl-2 mRNA by the SXL-UNR 3' UTR corepressor complex, which inhibits ribosome recruitment by an unknown mechanism. Here, we reveal a key role for the poly(A)-binding protein (PABP), a translational activator, in this inhibitory mechanism. Efficient msl-2 mRNA silencing via the 3' UTR requires both a poly(A) tail and PABP function, and we find that UNR directly interacts with PABP. To investigate how the repressor complex and PABP affect RNP composition during early steps in translation initiation, we established direct biochemical assays for synergistic recruitment of eIF4F and ribosomes by the cap and poly(A) tail. We find that the repressor complex targets ribosome binding after PABP-mediated recruitment of eIF4E/G. Our results uncover an important regulatory mechanism of Drosophila dosage compensation and provide insight into PABP-dependent translational control by 3' UTR-bound regulatory proteins.
Drosophila miR2 primarily targets the m7GpppN cap structure for translational repression.
Zdanowicz, A., Thermann, R., Kowalska, J., Jemielity, J., Duncan, K., Preiss, T., Darzynkiewicz, E. & Hentze, M.W.
Mol Cell. 2009 Sep 24;35(6):881-8.
Understanding the molecular mechanism(s) of how miRNAs repress mRNA translation is a fundamental challenge in RNA biology. Here we use a validated cell-free system from Drosophila embryos to investigate how miR2 inhibits translation initiation. By screening a library of chemical m7GpppN cap structure analogs, we identified defined modifications of the triphosphate backbone that augment miRNA-mediated inhibition of translation initiation but are "neutral" toward general cap-dependent translation. Interestingly, these caps also augment inhibition by 4E-BP. Kinetic dissection of translational repression and miR2-induced deadenylation shows that both processes proceed largely independently, with establishment of the repressed state involving a slow step. Our data demonstrate a primary role for the m7GpppN cap structure in miRNA-mediated translational inhibition, implicate structural determinants outside the core eIF4E-binding region in this process, and suggest that miRNAs may target cap-dependent translation through a mechanism related to the 4E-BP class of translational regulators.
In vivo role(s) of the iron regulatory proteins (IRP) 1 and 2 in aseptic local inflammation.
Viatte, L., Grone, H.J., Hentze, M.W. & Galy, B.
J Mol Med. 2009 Sep;87(9):913-21. Epub 2009 Jun 17.
The maintenance of iron homeostasis is critical as both iron deficiency and iron excess are deleterious. In mammals, iron homeostasis is regulated systemically by the iron-hormone hepcidin, an acute-phase protein secreted by the liver which inhibits iron absorption and recycling. Cellularly, the interaction of iron regulatory proteins (IRP) 1 and 2 with iron-responsive elements controls the expression of target mRNAs encoding proteins of iron acquisition, storage, utilization, and export. These processes critically affect iron levels, which in turn impact on numerous aspects of inflammation. To explore the role of IRP1 and IRP2 in inflammation, IRP-deficient mice, i.e., mice with total and constitutive deficiency of either IRP, were subjected to acute aseptic local inflammation. Turpentine oil injection increases the expression of acute phase proteins in the liver and interleukin 6 levels in the serum of control mice. Both IRP-deficient mouse models mount the same responses, indicating that the treatment was efficient in all animals and that the acute phase response does not require expression of both IRPs. As expected, turpentine oil treatment enhances hepcidin mRNA expression in the liver of wild-type mice, associated with decreased serum iron levels. Importantly, Irp1 (-/-) and Irp2 (-/-) animals, respectively, display quantitatively similar hepcidin mRNA induction and the appropriate reduction of the serum iron values. Our data indicate that the response of Irp1 (-/-) and Irp2 (-/-) mice to acute local inflammation is largely preserved.
Unusual bipartite mode of interaction between the nonsense-mediated decay factors, UPF1 and UPF2.
Clerici, M., Mourao, A., Gutsche, I., Gehring, N.H., Hentze, M.W., Kulozik, A., Kadlec, J., Sattler, M. & Cusack, S.
EMBO J. 2009 Jun 25;28(15):2293-2306.
Nonsense-mediated decay (NMD) is a eukaryotic quality control mechanism that degrades mRNAs carrying premature stop codons. In mammalian cells, NMD is triggered when UPF2 bound to UPF3 on a downstream exon junction complex interacts with UPF1 bound to a stalled ribosome. We report structural studies on the interaction between the C-terminal region of UPF2 and intact UPF1. Crystal structures, confirmed by EM and SAXS, show that the UPF1 CH-domain is docked onto its helicase domain in a fixed configuration. The C-terminal region of UPF2 is natively unfolded but binds through separated alpha-helical and beta-hairpin elements to the UPF1 CH-domain. The alpha-helical region binds sixfold more weakly than the beta-hairpin, whereas the combined elements bind 80-fold more tightly. Cellular assays show that NMD is severely affected by mutations disrupting the beta-hairpin binding, but not by those only affecting alpha-helix binding. We propose that the bipartite mode of UPF2 binding to UPF1 brings the ribosome and the EJC in close proximity by forming a tight complex after an initial weak encounter with either element.
The hierarchy of exon-junction complex assembly by the spliceosome explains key features of mammalian nonsense-mediated mRNA decay.
Gehring, N.H., Lamprinaki, S., Hentze, M.W. & Kulozik, A.E.
PLoS Biol. 2009 May 26;7(5):e1000120. Epub 2009 May 26.
Exon junction complexes (EJCs) link nuclear splicing to key features of mRNA function including mRNA stability, translation, and localization. We analyzed the formation of EJCs by the spliceosome, the physiological EJC assembly machinery. We studied a comprehensive set of eIF4A3, MAGOH, and BTZ mutants in complete or C-complex-arrested splicing reactions and identified essential interactions of EJC proteins during and after EJC assembly. These data establish that EJC deposition proceeds through a defined intermediate, the pre-EJC, as an ordered, sequential process that is coordinated by splicing. The pre-EJC consists of eIF4A3 and MAGOH-Y14, is formed before exon ligation, and provides a binding platform for peripheral EJC components that join after release from the spliceosome and connect the core structure with function. Specifically, we identified BTZ to bridge the EJC to the nonsense-mediated messenger RNA (mRNA) decay protein UPF1, uncovering a critical link between mRNP architecture and mRNA stability. Based on this systematic analysis of EJC assembly by the spliceosome, we propose a model of how a functional EJC is assembled in a strictly sequential and hierarchical fashion, including nuclear splicing-dependent and cytoplasmic steps.
Disassembly of exon junction complexes by PYM.
Gehring, N.H., Lamprinaki, S., Kulozik, A.E. & Hentze, M.W.
Cell. 2009 May 1;137(3):536-48.
Exon junction complexes (EJCs) are deposited onto mRNAs during splicing, serve as positional landmarks for the intron exon structure of genes, and direct posttranscriptional processes in the cytoplasm. EJC removal and recycling by translation are ill understood and have been attributed to ribosomal passage. This work identifies the ribosome-associated protein PYM as an EJC disassembly factor and defines its mechanism of function. Whereas EJC assembly intermediates are resistant to PYM, fully assembled EJCs are dissociated from spliced mRNAs by PYM. This disassembly involves PYM binding to the EJC proteins MAGOH-Y14. PYM overexpression in cells disrupts EJC association with spliced mRNA and inhibits nonsense-mediated mRNA decay. In cells depleted of PYM, EJCs accumulate on spliced mRNAs and EJC protein recycling is impaired. Hence, PYM is an EJC disassembly factor that acts both in vitro and in living cells, and that antagonizes important EJC functions.
Bone morphogenetic protein (BMP)-responsive elements located in the proximal and distal hepcidin promoter are critical for its response to HJV/BMP/SMAD.
Casanovas, G., Mleczko-Sanecka, K., Altamura, S., Hentze, M.W. & Muckenthaler, M.U.
J Mol Med. 2009 May;87(5):471-80. Epub 2009 Feb 20.
The hemochromatosis proteins HFE, transferrin receptor 2 (TfR2) and hemojuvelin (HJV, HFE2) positively control expression of the major iron regulatory hormone hepcidin. HJV is a bone morphogenetic protein (BMP) co-receptor that enhances the cellular response to BMP cytokines via the phosphorylation of SMAD proteins. In this study, we show that two highly conserved and sequence-identical BMP-responsive elements located at positions -84/-79 (BMP-RE1) and -2,255/-2,250 (BMP-RE2) of the human hepcidin promoter are critical for both the basal hepcidin mRNA expression and the hepcidin response to BMP-2 and BMP-6. While BMP-RE1 and BMP-RE2 show additive effects in responding to HJV-mediated BMP signals, only BMP-RE1 that is located in close proximity to a previously identified STAT-binding site is important for the hepcidin response to IL-6. These data identify a missing link between the HJV/BMP signaling pathways and hepcidin transcription, and further define the connection between inflammation and BMP-dependent hepcidin promoter activation. As such, they provide important new information furthering our understanding of disorders of iron metabolism and the anemia of inflammation.
Cell-autonomous and systemic context-dependent functions of iron regulatory protein 2 in mammalian iron metabolism.
Ferring-Appel, D., Hentze, M.W. & Galy, B.
Blood. 2009 Jan 15;113(3):679-87.
Mice with total and constitutive iron regulatory protein 2 (IRP2) deficiency exhibit microcytosis and altered body iron distribution with duodenal and hepatic iron loading and decreased iron levels in splenic macrophages. To explore cell-autonomous and systemic context-dependent functions of IRP2 and to assess the systemic consequences of local IRP2 deficiency, we applied Cre/Lox technology to specifically ablate IRP2 in enterocytes, hepatocytes, or macrophages, respectively. This study reveals that the hepatic and duodenal manifestations of systemic IRP2 deficiency are largely explained by cell-autonomous functions of IRP2. By contrast, IRP2-deficient macrophages from otherwise IRP2-sufficient mice do not display the abnormalities of macrophages from systemically IRP2-deficient animals, suggesting that these result from IRP2 disruption in other cell type(s). Mice with enterocyte-, hepatocyte-, or macrophage-specific IRP2 deficiency display normal red blood cell and plasma iron parameters, supporting the notion that the microcytosis in IRP2-deficient mice likely reflects an intrinsic defect in hematopoiesis. This work defines the respective roles of IRP2 in the determination of critical body iron parameters such as organ iron loading and erythropoiesis.
Translation initiation by the c-myc mRNA internal ribosome entry sequence and the poly(A) tail.
Thoma, C., Fraterman, S., Gentzel, M., Wilm, M. & Hentze, M.W.
RNA. 2008 Aug;14(8):1579-89. Epub 2008 Jun 12.
Eukaryotic mRNAs possess a poly(A) tail that enhances translation via the (7)mGpppN cap structure or internal ribosome entry sequences (IRESs). Here we address the question of how cellular IRESs recruit the ribosome and how recruitment is augmented by the poly(A) tail. We show that the poly(A) tail enhances 48S complex assembly by the c-myc IRES. Remarkably, this process is independent of the poly(A) binding protein (PABP). Purification of native 48S initiation complexes assembled on c-myc IRES mRNAs and quantitative label-free analysis by liquid chromatography and mass spectrometry directly identify eIFs 2, 3, 4A, 4B, 4GI, and 5 as components of the c-myc IRES 48S initiation complex. Our results demonstrate for the first time that the poly(A) tail augments the initiation step of cellular IRES-driven translation and implicate a distinct subset of translation initiation factors in this process. The mechanistic distinctions from cap-dependent translation may allow specific translational control of the c-myc mRNA and possibly other cellular mRNAs that initiate translation via IRESs.
A bone morphogenetic protein (BMP)-responsive element in the hepcidin promoter controls HFE2-mediated hepatic hepcidin expression and its response to IL-6 in cultured cells.
Verga Falzacappa, M.V., Casanovas, G., Hentze, M.W. & Muckenthaler, M.U.
J Mol Med (Berl). 2008 May;86(5):531-40. doi: 10.1007/s00109-008-0313-7. Epub2008 Apr 18.
The precise regulation of the iron-regulatory hormone hepcidin is essential to maintain body iron homeostasis: Hepcidin deficiency induces iron overload, and hepcidin excess results in anaemia. Mutations in the gene HFE2 cause severe iron overload and are associated with low hepcidin expression. Recent data suggest that HFE2 is a bone morphogenetic protein (BMP) co-receptor, and that the decreased hepcidin mRNA expression because of HFE2 dysfunction is a result of impaired BMP signalling ability. In this study, we identify a critical BMP-responsive element (BMP-RE) at position -84/-79 of the hepcidin promoter. We show that this element mediates HFE2-dependent basal hepcidin mRNA expression under control conditions. Unexpectedly, the mutation of the same BMP-RE element also severely impairs hepcidin activation in response to IL-6. These data uncover a missing link in the HFE2-mediated control of hepcidin expression and suggest that the BMP-RE controls hepcidin promoter activity mediated by HFE2 and inflammatory stimuli.
Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways.
Ivanov, P.V., Gehring, N.H., Kunz, J.B., Hentze, M.W. & Kulozik, A.E.
EMBO J. 2008 Mar 5;27(5):736-47. Epub 2008 Feb 7.
Nonsense-mediated mRNA decay (NMD) represents a key mechanism to control the expression of wild-type and aberrant mRNAs. Phosphorylation of the protein UPF1 in the context of translation termination contributes to committing mRNAs to NMD. We report that translation termination is inhibited by UPF1 and stimulated by cytoplasmic poly(A)-binding protein (PABPC1). UPF1 binds to eRF1 and to the GTPase domain of eRF3 both in its GTP- and GDP-bound states. Importantly, mutation studies show that UPF1 can interact with the exon junction complex (EJC) alternatively through either UPF2 or UPF3b to become phosphorylated and to activate NMD. On this basis, we discuss an integrated model where UPF1 halts translation termination and is phosphorylated by SMG1 if the termination-promoting interaction of PABPC1 with eRF3 cannot readily occur. The EJC, with UPF2 or UPF3b as a cofactor, interferes with physiological termination through UPF1. This model integrates previously competing models of NMD and suggests a mechanistic basis for alternative NMD pathways.
Hfe acts in hepatocytes to prevent hemochromatosis.
Vujic Spasic, M., Kiss, J., Herrmann, T., Galy, B., Martinache, S., Stolte, J., Grone, H.J., Stremmel, W., Hentze, M.W. & Muckenthaler, M.U.
Cell Metab. 2008 Feb;7(2):173-8.
Hereditary hemochromatosis (HH) is a prevalent, potentially fatal disorder of iron metabolism hallmarked by intestinal hyperabsorption of iron, hyperferremia, and hepatic iron overload. In both humans and mice, type I HH is associated with mutations in the broadly expressed HFE/Hfe gene. To identify where Hfe acts to prevent HH, we generated mice with tissue-specific Hfe ablations. This work demonstrates that local Hfe expression in hepatocytes serves to maintain physiological iron homeostasis, answering a long-standing question in medicine and explaining earlier clinical observations.
Iron regulatory proteins are essential for intestinal function and control key iron absorption molecules in the duodenum.
Galy, B., Ferring-Appel, D., Kaden, S., Grone, H.J. & Hentze, M.W.
Cell Metab. 2008 Jan;7(1):79-85.
Iron regulatory proteins (IRPs) orchestrate the posttranscriptional regulation of critical iron metabolism proteins at the cellular level. Redundancy between IRP1 and IRP2 associated with embryonic lethality of doubly IRP-deficient mice has precluded the study of IRP function in vivo. Here we use Cre/Lox technology to generate viable organisms lacking IRP expression in a single tissue, the intestine. Mice lacking intestinal IRP expression develop intestinal malabsorption and dehydration postnatally and die within 4 weeks of birth. We demonstrate that IRPs control the expression of divalent metal transporter 1 (DMT1) mRNA and protein, a limiting intestinal iron importer. IRPs are also shown to be critically important to secure physiological levels of the basolateral iron exporter ferroportin. IRPs are thus essential for intestinal function and organismal survival and coordinate the synthesis of key iron metabolism proteins in the duodenum.
Systemic iron homeostasis and the iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network.
Muckenthaler, M.U., Galy, B. & Hentze, M.W.
Annu Rev Nutr. 2008;28:197-213.
The regulation and maintenance of systemic iron homeostasis is critical to human health. Iron overload and deficiency diseases belong to the most common nutrition-related pathologies across the globe. It is now well appreciated that the hormonal hepcidin/ferroportin system plays an important regulatory role for systemic iron metabolism. We review recent data that uncover the importance of the cellular iron-responsive element/iron-regulatory protein (IRE/IRP) regulatory network in systemic iron homeostasis. We also discuss how the IRE/IRP regulatory system communicates with the hepcidin/ferroportin system to connect the control networks for systemic and cellular iron balance.
Tethering assays to investigate nonsense-mediated mRNA decay activating proteins.
Gehring, N.H., Hentze, M.W. & Kulozik, A.E.
Methods Enzymol. 2008;448:467-82.
Nonsense-mediated mRNA decay (NMD) is activated by exon-junction complexes (EJCs) that are located downstream of the termination codon of the substrate mRNAs. This situation can be imitated by tethering components of the EJC to the 3' untranslated region (3' UTR) of a reporter mRNA. Here we describe the detailed use of two analogous tethering systems that are based on the coat protein of bacteriophage MS2 or on the 22 amino acid RNA-binding domain of the bacteriophage lambda-antiterminator protein N (lambdaN-peptide). These polypeptides are fused as tags to proteins of interest. Their respective RNA binding sites are inserted into reporter mRNAs. This enables recruitment of the NMD activity of the fusion protein to an NMD-activating position, bypassing the requirement for splicing. In this chapter we explicate the cloning of appropriate reporter plasmids and the setup of a tethering experiment with the necessary control experiments. Advantages of the different systems and tags are discussed.
miChip: an array-based method for microRNA expression profiling using locked nucleic acid capture probes.
Castoldi, M., Schmidt, S., Benes, V., Hentze, M.W. & Muckenthaler, M.U.
Nat Protoc. 2008;3(2):321-9.
MicroRNAs (miRNAs) represent a class of short (22 nt) noncoding RNAs that control gene expression post-transcriptionally. Microarray technology is frequently applied to monitor miRNA expression levels but is challenged by (i) the short length of miRNAs that offers little sequence for appending detection molecules; (ii) low copy number of some miRNA; and (iii) a wide range of predicted melting temperatures (Tm) versus their DNA complementary sequences. We recently developed a microarray platform for genome-wide profiling of miRNAs (miChip) by applying locked nucleic acid (LNA)-modified capture probes. Here, we provide detailed protocols for the generation of the miChip microarray platform, the preparation and fluorescent labeling of small RNA containing total RNA, its hybridization to the immobilized LNA-modified capture probes and the post-hybridization handling of the microarray. Starting from the intact tissue sample, the entire protocol takes approximately 3 d to yield highly accurate and sensitive data about miRNA expression levels.
A conserved motif in Argonaute-interacting proteins mediates functional interactions through the Argonaute PIWI domain.
Till, S., Lejeune, E., Thermann, R., Bortfeld, M., Hothorn, M., Enderle, D., Heinrich, C., Hentze, M.W. & Ladurner, A.G.
Nat Struct Mol Biol. 2007 Oct;14(10):897-903. Epub 2007 Sep 23.
Argonaute (Ago) proteins mediate silencing of nucleic acid targets by small RNAs. In fission yeast, Ago1, Tas3 and Chp1 assemble into a RITS complex, which silences transcription near centromeres. Here we describe a repetitive motif within Tas3, termed the 'Argonaute hook', that is conserved from yeast to humans and binds Ago proteins through their PIWI domains in vitro and in vivo. Site-directed mutation of key residues in the motif disrupts Ago binding and heterochromatic silencing in vivo. Unexpectedly, a PIWI domain pocket that binds the 5' end of the short interfering RNA guide strand is required for direct binding of the Ago hook. Moreover, wild-type but not mutant Ago hook peptides derepress microRNA-mediated translational silencing of a target messenger RNA. Proteins containing the conserved Ago hook may thus be important regulatory components of effector complexes in RNA interference.
miChip: a microarray platform for expression profiling of microRNAs based on locked nucleic acid (LNA) oligonucleotide capture probes.
Castoldi, M., Benes, V., Hentze, M.W. & Muckenthaler, M.U.
Methods. 2007 Oct;43(2):146-52.
As key regulators of post-transcriptional gene expression, it is important to monitor the expression of microRNAs (miRNA) in diverse physiological and pathophysiological processes. Here, we describe a method for sensitive and accurate microarray-based expression profiling of miRNAs. The protocol focuses on the use of locked nucleic acid (LNA)-modified capture probes. LNAs are bicyclic nucleotide analogues that significantly increase the melting temperature (T(m)) of hybrids with miRNAs. Mixed LNA/DNA capture probes thus can be designed for equal T(m)s for all miRNAs, which naturally cover a range between 45 and 74 degrees C. The protocols established are easy to apply, as they do not require RNA size selection and/or amplification of miRNAs. Moreover, they enable high affinity hybridizations yielding accurate signals that discriminate between single nucleotide differences and hence closely related miRNA family members.
Identification of target mRNAs of regulatory RNA-binding proteins using mRNP immunopurification and microarrays.
Sanchez, M., Galy, B., Hentze, M.W. & Muckenthaler, M.U.
Nat Protoc. 2007 Aug;2(8):2033-2042.
RNA-binding proteins (RBPs) frequently regulate the post-transcriptional fate of target mRNAs. To identify novel target mRNAs of RBPs, we incubate total RNA with recombinant RBP and immunoselect the messenger-ribonucleoproteins using a specific anti-RBP antibody. The mRNA composition of the supernatant and/or immunoprecipitated fraction is analyzed using dual-color microarrays in comparison with control reaction. From start to finish, the protocol takes approximately 6 d.
Expression of the subgenomic hepatitis C virus replicon alters iron homeostasis in Huh7 cells.
Fillebeen, C., Muckenthaler, M., Andriopoulos, B., Bisaillon, M., Mounir, Z., Hentze, M.W., Koromilas, A.E. & Pantopoulos, K.
J Hepatol. 2007 Jul;47(1):12-22. Epub 2007 Mar 2.
BACKGROUND/AIMS: Infection with hepatitis C virus (HCV) is associated with alterations in body iron homeostasis by poorly defined mechanisms. To seek for molecular links, we employed an established cell culture model for viral replication, and assessed how the expression of an HCV subgenomic replicon affects iron metabolism in host Huh7 hepatoma cells. METHODS: The expression of iron metabolism genes and parameters defining the cellular iron status were analyzed and compared between parent and replicon Huh7 cells. RESULTS: By using the IronChip microarray platform, we observed replicon-induced changes in expression profiles of iron metabolism genes. Notably, ceruloplasmin mRNA and protein expression were decreased in replicon cells. In addition, transferrin receptor 1 (TfR1) was also downregulated, while ferroportin levels were elevated, resulting in reduced iron uptake and increased iron release capacity of replicon cells. These responses were associated with an iron-deficient phenotype, manifested in decreased levels of the "labile iron pool" and concomitant induction of IRE-binding activity and IRP2 expression. Furthermore, hemin-treated replicon cells exhibited a defect in retaining iron. The clearance of the replicon by prolonged treatment with interferon-alpha only partially reversed the iron-deficient phenotype but almost completely restored the capacity of cured cells to retain iron. CONCLUSIONS: We propose that Huh7 cells undergo genetic reprogramming to permit subgenomic viral replication that results in reduction of intracellular iron levels. This response may provide a mechanism to bypass iron-mediated inactivation of the viral RNA polymerase NS5B.
Splicing factors stimulate polyadenylation via USEs at non-canonical 3' end formation signals.
Danckwardt, S., Kaufmann, I., Gentzel, M., Foerstner, K.U., Gantzert, A.S., Gehring, N.H., Neu-Yilik, G., Bork, P., Keller, W., Wilm, M., Hentze, M.W. & Kulozik, A.E.
EMBO J. 2007 Jun 6;26(11):2658-69. Epub 2007 Apr 26.
The prothrombin (F2) 3' end formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3' UTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of USE function. We show that the F2 USE contains a highly conserved nonameric core sequence, which promotes 3' end formation in a position- and sequence-dependent manner. We identify proteins that specifically interact with the USE, and demonstrate their function as trans-acting factors that promote 3' end formation. Interestingly, these include the splicing factors U2AF35, U2AF65 and hnRNPI. We show that these splicing factors not only modulate 3' end formation via the USEs contained in the F2 and the complement C2 mRNAs, but also in the biocomputationally identified BCL2L2, IVNS and ACTR mRNAs, suggesting a broader functional role. These data uncover a novel mechanism that functionally links the splicing and 3' end formation machineries of multiple cellular mRNAs in an USE-dependent manner.
Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation.
Thermann, R. & Hentze, M.W.
Nature. 2007 Jun 14;447(7146):875-8. Epub 2007 May 16.
MicroRNAs (miRs) inhibit protein synthesis by mechanisms that are as yet unresolved. We developed a cell-free system from Drosophila melanogaster embryos that faithfully recapitulates miR2-mediated translational control by means of the 3' untranslated region of the D. melanogaster reaper messenger RNA. Here we show that miR2 inhibits translation initiation without affecting mRNA stability. Surprisingly, miR2 induces the formation of dense (heavier than 80S) miRNPs ('pseudo-polysomes') even when polyribosome formation and 60S ribosomal subunit joining are blocked. An mRNA bearing an ApppG instead of an m7GpppG cap structure escapes the miR2-mediated translational block. These results directly show the inhibition of m7GpppG cap-mediated translation initiation as the mechanism of miR2 function, and uncover pseudo-polysomal messenger ribonucleoprotein assemblies that may help to explain earlier findings.
Omeprazole decreases duodenal iron absorption via inhibition of DMT-1 function.
Mair, SM; Ludwiczek, S; Muckenthaler, M; Schwelberger, H; Theurl, I; Weiss, H; Hentze, M.W.; Ritter, M; Weiss, G
American Journal of Hematology June 2007 82(6)
MiRNA expression in T-cell acute lymphoblastic leukaemia
Kox, C; Breit, S; Castoldi, M; Kirschner-Schwabe, R; Ludwig, WD; Hentze, M.W.; Kulozik, AE; Muckenthaler, MU
Klinische Pädiatrie June 2007 219(3)
Metal distribution and gene expression profile in DMT1 knockout mice
Gunshin, H; Tohyama, C; Vainshtein, Y; Hentze, M.W.; Muckenthaler, M; Andrews, NC
American Journal of Hematology June 2007 82(6)
Iron-regulatory proteins limit hypoxia-inducible factor-2alpha expression in iron deficiency.
Sanchez, M., Galy, B., Muckenthaler, M.U. & Hentze, M.W.
Nat Struct Mol Biol. 2007 May;14(5):420-6. Epub 2007 Apr 8.
Hypoxia stimulates erythropoiesis, the major iron-utilization pathway. We report the discovery of a conserved, functional iron-responsive element (IRE) in the 5' untranslated region of the messenger RNA encoding endothelial PAS domain protein-1, EPAS1 (also called hypoxia-inducible factor-2alpha, HIF2alpha). Via this IRE, iron regulatory protein binding controls EPAS1 mRNA translation in response to cellular iron availability. Our results uncover a regulatory link that permits feedback control between iron availability and the expression of a key transcription factor promoting iron utilization. They also show that an IRE that is structurally distinct from, for example, the ferritin mRNA IRE and that has been missed by in silico approaches, can mediate mechanistically similar responses.
Physiologic systemic iron metabolism in mice deficient for duodenal Hfe.
Spasic, M.V., Kiss, J., Herrmann, T., Kessler, R., Stolte, J., Galy, B., Rathkolb, B., Wolf, E., Stremmel, W., Hentze, M.W. & Muckenthaler, M.U.
Blood. 2007 May 15;109(10):4511-7. Epub 2007 Jan 30.
Mutations in the Hfe gene result in hereditary hemochromatosis (HH), a disorder characterized by increased duodenal iron absorption and tissue iron overload. Identification of a direct interaction between Hfe and transferrin receptor 1 in duodenal cells led to the hypothesis that the lack of functional Hfe in the duodenum affects TfR1-mediated serosal uptake of iron and misprogramming of the iron absorptive cells. Contrasting this view, Hfe deficiency causes inappropriately low expression of the hepatic iron hormone hepcidin, which causes increased duodenal iron absorption. We specifically ablated Hfe expression in mouse enterocytes using Cre/LoxP technology. Mice with efficient deletion of Hfe in crypt- and villi-enterocytes maintain physiologic iron metabolism with wild-type unsaturated iron binding capacity, hepatic iron levels, and hepcidin mRNA expression. Furthermore, the expression of genes encoding the major intestinal iron transporters is unchanged in duodenal Hfe-deficient mice. Our data demonstrate that intestinal Hfe is dispensable for the physiologic control of systemic iron homeostasis under steady state conditions. These findings exclude a primary role for duodenal Hfe in the pathogenesis of HH and support the model according to which Hfe is required for appropriate expression of the "iron hormone" hepcidin which then controls intestinal iron absorption.
Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1.
Ludwiczek, S., Theurl, I., Muckenthaler, M.U., Jakab, M., Mair, S.M., Theurl, M., Kiss, J., Paulmichl, M., Hentze, M.W., Ritter, M. & Weiss, G.
Nat Med. 2007 Apr;13(4):448-54. Epub 2007 Feb 11.
Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.
STAT3 mediates hepatic hepcidin expression and its inflammatory stimulation.
Verga Falzacappa, M.V., Vujic Spasic, M., Kessler, R., Stolte, J., Hentze, M.W. & Muckenthaler, M.U.
Blood. 2007 Jan 1;109(1):353-8. Epub 2006 Aug 31.
Hepcidin is a key iron-regulatory hormone produced by the liver. Inappropriately low hepcidin levels cause iron overload, while increased hepcidin expression plays an important role in the anemia of inflammation (AI) by restricting intestinal iron absorption and macrophage iron release. Its expression is modulated in response to body iron stores, hypoxia, and inflammatory and infectious stimuli involving at least in part cytokines secreted by macrophages. In this study we established and characterized IL6-mediated hepcidin activation in the human liver cell line Huh7. We show that the proximal 165 bp of the hepcidin promoter is critical for hepcidin activation in response to exogenously administered IL6 or to conditioned medium from the monocyte/macrophage cell line THP-1. Importantly, we show that hepcidin activation by these stimuli requires a STAT3 binding motif located at position -64/-72 of the promoter. The same STAT binding site is also required for high basal-level hepcidin mRNA expression under control culture conditions, and siRNA-mediated RNA knockdown of STAT3 strongly reduces hepcidin mRNA expression. These results identify a missing link in the acute-phase activation of hepcidin and establish STAT3 as a key effector of baseline hepcidin expression and during inflammatory conditions.
The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway.
Viegas, M.H., Gehring, N.H., Breit, S., Hentze, M.W. & Kulozik, A.E.
Nucleic Acids Res. 2007;35(13):4542-51. Epub 2007 Jun 22.
Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. NMD has been shown to also regulate normal gene expression and thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Recently, NMD efficiency has been shown to vary between cell types and individuals thus implicating NMD as a modulator of genetic disease severity. We have now specifically analysed the molecular mechanism of variable NMD efficiency and first established an assay system for the quantification of NMD efficiency, which is based on carefully validated cellular NMD target transcripts. In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains. In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model. We conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that cell type specific co-factor availability represents a novel principle that controls NMD.
Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5' untranslated region.
Mihailovich, M., Thermann, R., Grohovaz, F., Hentze, M.W. & Zacchetti, D.
Nucleic Acids Res. 2007;35(9):2975-85. Epub 2007 Apr 16.
BACE1 is the protease responsible for the production of amyloid-beta peptides that accumulate in the brain of Alzheimer's disease (AD) patients. BACE1 expression is regulated at the transcriptional, as well as post-transcriptional level. Very high BACE1 mRNA levels have been observed in pancreas, but the protein and activity were found mainly in brain. An up-regulation of the protein has been described in some AD patients without a change in transcript levels. The features of BACE1 5' untranslated region (5' UTR), such as the length, GC content, evolutionary conservation and presence of upstream AUGs (uAUGs), indicate an important regulatory role of this 5' UTR in translational control. We demonstrate that, in brain and pancreas, almost all of the native BACE1 mRNA contains the full-length 5' UTR. RNA transfection and in vitro translation show that translation is mainly inhibited by the presence of the uAUGs. We provide a mutational analysis that highlight the second uAUG as the main inhibitory element while mutations of all four uAUGs fully de-repress translation. Furthermore, we have evidence that a sequence within the region 222-323 of the BACE1 5' UTR has a stimulatory effect on translation that might depend on the presence of trans-acting factors.
Studying translational control in Drosophila cell-free systems.
Gebauer, F. & Hentze, M.W.
Methods Enzymol. 2007;429:23-33.
Classically, Drosophila cell-free translation systems have been used to study the response of the translational machinery to heat shock treatment. We and others have developed optimized Drosophila embryo and ovary extracts, and their use has expanded to the study of a variety of translational control events. These extracts recapitulate many of the aspects of mRNA translation observed in vivo and retain critical regulatory features of several translational control processes. Indeed, their use is rapidly improving our knowledge of molecular mechanisms of translational control. In this chapter we provide general guidelines and detailed protocols to obtain and use translation extracts derived from Drosophila embryos and ovaries.
A chemiluminescence-based reporter system to monitor nonsense-mediated mRNA decay.
Boelz, S., Neu-Yilik, G., Gehring, N.H., Hentze, M.W. & Kulozik, A.E.
Biochem Biophys Res Commun. 2006 Oct 13;349(1):186-91. Epub 2006 Aug 11.
Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that mediates rapid degradation of transcripts bearing premature translation termination codons (PTCs) and thereby limits the expression of unproductively processed mRNAs and the synthesis of C-terminally truncated peptides. Both its importance as a means to control gene expression and in the context of genetic and acquired human diseases call for an exploration of the mammalian NMD pathway using chemical biology approaches. Here, we describe a novel cell-based chemiluminescence reporter system that recapitulates the hallmark features of mammalian NMD. The assay is characterized by its high sensitivity, robustness, and its potential for automated handling. Limiting NMD efficiency by RNAi-mediated depletion of the essential NMD factor UPF1 markedly and specifically increased the NMD reporter mRNA level and resulted in a proportional increase in protein expression reflected by Renilla luminescence. The PI 3-kinase inhibitor wortmannin has previously been found to up-modulate PTC-containing transcripts by inhibiting the UPF1 kinase SMG1. Wortmannin treatment enhanced NMD reporter expression in our system in a dose-dependent way, illustrating its utility for small molecule screening.
Iron homeostasis in the brain: complete iron regulatory protein 2 deficiency without symptomatic neurodegeneration in the mouse.
Galy, B., Holter, S.M., Klopstock, T., Ferring, D., Becker, L., Kaden, S., Wurst, W., Grone, H.J. & Hentze, M.W.
Nat Genet. 2006 Sep;38(9):967-9. Europe PMC
Iron regulation and the cell cycle: identification of an iron-responsive element in the 3'-untranslated region of human cell division cycle 14A mRNA by a refined microarray-based screening strategy.
Sanchez, M., Galy, B., Dandekar, T., Bengert, P., Vainshtein, Y., Stolte, J., Muckenthaler, M.U. & Hentze, M.W.
J Biol Chem. 2006 Aug 11;281(32):22865-74. Epub 2006 Jun 7.
Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5'- or 3'-untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. IRP/IRE messenger ribonucleoproteins were immunoselected, and their mRNA composition was analyzed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Among different candidates, this report focuses on a novel IRE located in the 3'-untranslated region of the cell division cycle 14A mRNA. We show that this IRE motif efficiently binds both IRP1 and IRP2. Differential splicing of cell division cycle 14A produces IRE- and non-IRE-containing mRNA isoforms. Interestingly, only the expression of the IRE-containing mRNA isoforms is selectively increased by cellular iron deficiency. This work describes a new experimental strategy to explore the IRE/IRP regulatory network and uncovers a previously unrecognized regulatory link between iron metabolism and the cell cycle.
Internal ribosome entry sequence-mediated translation initiation triggers nonsense-mediated decay.
Holbrook, J.A., Neu-Yilik, G., Gehring, N.H., Kulozik, A.E. & Hentze, M.W.
EMBO Rep. 2006 Jul;7(7):722-726. Epub 2006 Jun 16.
In eukaryotes, a surveillance pathway known as nonsense-mediated decay (NMD) regulates the abundance of messenger RNAs containing premature termination codons (PTCs). In mammalian cells, it has been asserted that the NMD-relevant first round of translation is special and involves initiation by a specific protein heterodimer, the nuclear cap-binding complex (CBC). Arguing against a requirement for CBC-mediated translation initiation, we show that ribosomal recruitment by the internal ribosomal entry sequence of the encephalomyocarditis virus triggers NMD of a PTC-containing transcript under conditions in which ribosome entry from the cap is prohibited. These data generalize the previous model and suggest that translation per se, irrespective of how it is initiated, can mediate NMD.
IRP1-independent alterations of cardiac iron metabolism in doxorubicin-treated mice.
Corna, G., Galy, B., Hentze, M.W. & Cairo, G.
J Mol Med. 2006 Jul;84(7):551-60. Epub 2006 Jun 13.
Iron aggravates the cardiotoxicity of doxorubicin (DOX), a widely used anticancer anthracycline. The amount of iron in the cell is regulated by the iron regulatory proteins (IRPs)-1 and -2 that control the posttranscriptional expression of key iron metabolism genes. In vitro and cell culture studies revealed the ability of DOX to modulate the activity of both IRPs. However, conflicting data were obtained from different cell types and experimental conditions. To investigate the connection between acute DOX cardiotoxicity and the IRPs in a mammalian organism, we analyzed IRP activity and the expression of IRP target genes in the heart of mice subjected to DOX treatment. DOX exposure elicits a differential modulation of the two IRPs with reduced IRP2 activity and unchanged IRP1 activity. IRP2 downmodulation is associated with the upregulation of the ferritin L and H genes and decreased expression of the transferrin receptor 1 (TfR1). To directly test the role of IRP1 in DOX cardiotoxicity, the DOX response was analyzed in mice lacking IRP1. DOX-mediated IRP2 downmodulation and regulation of ferritin and TfR1 expression is identical in Irp1 (-/-) mice compared to wild type, as is the degree of oxidative damage of the heart assessed by thioredoxin and thiobarbituric acid reactive substance levels and by brain natriuretic peptide mRNA expression. These data demonstrate that the alterations of cardiac iron homeostasis related to acute anthracycline cardiotoxicity occur independently of IRP1. The observed IRP2 downmodulation could serve as a means to counteract DOX cardiotoxicity by reducing the "free" cellular iron pool.
Functions of hUpf3a and hUpf3b in nonsense-mediated mRNA decay and translation.
Kunz, J.B., Neu-Yilik, G., Hentze, M.W., Kulozik, A.E. & Gehring, N.H.
RNA. 2006 Jun;12(6):1015-22. Epub 2006 Apr 6.
The exon-junction complex (EJC) components hUpf3a and hUpf3b serve a dual function: They promote nonsense-mediated mRNA decay (NMD), and they also regulate translation efficiency. Whether these two functions are interdependent or independent of each other is unknown. We characterized the function of the hUpf3 proteins in a lambdaN/boxB-based tethering system. Despite the high degree of sequence similarity between hUpf3b and hUpf3a, hUpf3a is much less active than hUpf3b to induce NMD and to stimulate translation. We show that induction of NMD by hUpf3 proteins requires interaction with Y14, Magoh, BTZ, and eIF4AIII. The protein region that mediates this interaction and discriminates between hUpf3a and hUpf3b in NMD function is located in the C-terminal domain and fully contained within a small sequence that is highly conserved in Upf3b but not Upf3a proteins. Stimulation of translation is independent of this interaction and is determined by other regions of the hUpf3 protein, indicating the presence of different downstream pathways of hUpf3 proteins either in NMD or in translation.
The prothrombin 20209 C-->T mutation in Jewish-Moroccan Caucasians: molecular analysis of gain-of-function of 3' end processing.
Danckwardt, S., Hartmann, K., Katz, B., Hentze, M.W., Levy, Y., Eichele, R., Deutsch, V., Kulozik, A.E. & Ben-Tal, O.
J Thromb Haemost. 2006 May;4(5):1078-85.
BACKGROUND: Mutations of the 3' end mRNA-processing signal of the prothrombin (F2) gene have been reported to cause elevated F2 plasma concentrations, thrombosis, and complications of pregnancy. Whereas the common F2 20210*A mutation is almost exclusively found in Caucasians, the F2 20209*T mutation has been reported in Afro-Americans and Afro-Caribbeans only. PATIENTS AND METHODS: Using LightCycler technology, three unrelated Jewish-Moroccan patients tested for obstetric complications were found to be carriers of the F2 20209*T allele. A detailed molecular analysis was performed to identify the functional impact of this mutation. RESULTS: We report three unrelated women of Jewish-Moroccan origin with a F2 20209*T mutation and fetal loss or infertility. The functional analysis revealed that the F2 20209*T mutation stimulates 3' end processing and up-regulates prothrombin protein expression as assessed by a highly sensitive luminescence-based reporter system. CONCLUSIONS: This is the first report of 20209*T in Caucasians, and functional analysis demonstrates that F2 20209*T falls into a general category of mutations of the F2 gene, which may possibly contribute to thrombophilia and complications of pregnancy by interfering with a tightly balanced architecture of non-canonical F2 3' end formation signals.
A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).
Castoldi, M., Schmidt, S., Benes, V., Noerholm, M., Kulozik, A.E., Hentze, M.W. & Muckenthaler, M.U.
RNA. 2006 May;12(5):913-20. Epub 2006 Mar 15.
MicroRNAs represent a class of short (approximately 22 nt), noncoding regulatory RNAs involved in development, differentiation, and metabolism. We describe a novel microarray platform for genome-wide profiling of mature miRNAs (miChip) using locked nucleic acid (LNA)-modified capture probes. The biophysical properties of LNA were exploited to design probe sets for uniform, high-affinity hybridizations yielding highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. MiChip will greatly simplify miRNA expression profiling of biological and clinical samples.
The molecular circuitry regulating the switch between iron deficiency and overload in mice.
Mok, H., Mlodnicka, A.E., Hentze, M.W., Muckenthaler, M. & Schumacher, A.
J Biol Chem. 2006 Mar 24;281(12):7946-51. Epub 2006 Jan 17.
Recent positional cloning of the radiation-induced polycythaemia (Pcm) mutation revealed a 58-bp microdeletion in the promoter region of ferroportin 1 (Fpn1), the sole cellular iron exporter identified to date. Here we report a molecular definition of the regulatory mechanisms governing the dynamic changes in iron balance in Pcm heterozygous mice between 3 and 12 weeks of age. Hepatic and/or duodenal response patterns of iron metabolism genes, such as Trfr, cybrd1, and Slc11a2, explained the transition from early postnatal iron deficiency to iron overload by 12 weeks of age. A significant delay in developmental up-regulation of hepcidin (Hamp), the pivotal hormonal regulator of iron homeostasis, correlated with high levels of Fpn1 expression in hepatic Kupffer cells and duodenal epithelial cells at 7 weeks of age. Conversely, upon up-regulation of Hamp expression at 12 weeks of age, Fpn1 expression decreased, indicative of a Hamp-mediated homeostatic loop. Hamp regulation due to iron did not appear dependent on transcription-level changes of the murine homolog of Hemojuvelin (Rgmc). Aged cohorts of Pcm mice exhibited low levels of Fpn1 expression in the context of an iron-deficient erythropoiesis and profound iron sequestration in reticuloendothelial macrophages, duodenum, and other tissues. Thus, similar to the anemia of chronic disease, these findings demonstrate decreased iron bioavailability due to sustained down-regulation of Fpn1 levels by Hamp. We conclude that regulatory alleles, such as Pcm, with highly dynamic changes in iron balance are ideally suited to interrogate the genetic circuitry regulating iron metabolism.
Bruno acts as a dual repressor of oskar translation, promoting mRNA oligomerization and formation of silencing particles.
Chekulaeva, M., Hentze, M.W. & Ephrussi, A.
Cell. 2006 Feb 10;124(3):521-33.
Prior to reaching the posterior pole of the Drosophila oocyte, oskar mRNA is translationally silenced by Bruno binding to BREs in the 3' untranslated region. The eIF4E binding protein Cup interacts with Bruno and inhibits oskar translation. Validating current models, we directly demonstrate the mechanism proposed for Cup-mediated repression: inhibition of small ribosomal subunit recruitment to oskar mRNA. However, 43S complex recruitment remains inhibited in the absence of functional Cup, uncovering a second Bruno-dependent silencing mechanism. This mechanism involves mRNA oligomerization and formation of large (50S-80S) silencing particles that cannot be accessed by ribosomes. Bruno-dependent mRNA oligomerization into silencing particles emerges as a mode of translational control that may be particularly suited to coupling with mRNA transport.
Sex-lethal imparts a sex-specific function to UNR by recruiting it to the msl-2 mRNA 3' UTR: translational repression for dosage compensation.
Duncan, K., Grskovic, M., Strein, C., Beckmann, K., Niggeweg, R., Abaza, I., Gebauer, F., Wilm, M. & Hentze, M.W.
Genes Dev. 2006 Feb 1;20(3):368-79.
MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein sex-lethal (SXL), which binds to the msl-2 mRNA 5' and 3' untranslated regions (UTRs) and blocks translation through distinct UTR-specific mechanisms. Here, we purify translationally silenced msl-2 mRNPs and identify UNR (upstream of N-ras) as a protein recruited to the 3' UTR by SXL. We demonstrate that SXL requires UNR as a corepressor for 3'-UTR-mediated regulation, imparting a female-specific function to the ubiquitously expressed UNR protein. Our results reveal a novel functional role for UNR as a translational repressor and indicate that UNR is a key component of a "fail-safe" dosage compensation regulatory system that prevents toxic MSL-2 synthesis in female cells.
Targeted disruption of the mouse mitoferrin (Slc25A37) mitochondrial solute carrier results in defective primitive and definitive erythropoiesis.
Paw, Barry H.; Gwynn, Babette; Langer, Nathaniel B.; Shaw, George C.; Lambert, Amy J.; Boyer, Leah A.; Li, Liangtao; Stolte, Jens; Robledo, Raymond F.; Sahr, Kenneth E.; Schlaeger, Thorsten M.; Hentze, M.W.; Muckenthaler, Martina U.; Kaplan, Jerry; Peters, Luanne L.
Blood November 2006 108(11)
The physiology of prothrombin gene expression integrates RNA polyadenylation and splicing in a novel regulatable 3 ' RNP-complex.
Danckwardt, Sven; Gentzel, Marc; Gehring, Niels H.; Kaufmann, Isabelle; Neu-Yilik, Gabriele; Wilm, M.; Hentze, M.W.; Kulozjk, Andreas E.
Blood 2006 108(11)
3' end processing of the prothrombin mRNA in thrombophilia.
Danckwardt, S., Hartmann, K., Gehring, N.H., Hentze, M.W. & Kulozik, A.E.
Acta Haematol. 2006;115(3-4):192-7.
Clinical and genetic studies have led to the discovery of specific genotypes that predispose to thromboembolism in adults and children. The exploration of the underlying pathologies has revealed a broad variety of affected molecular mechanisms. Most recently, the functional analysis of the prothrombin (F2) 20210*A variant revealed increased efficiency of 3' end processing as a novel genetic mechanism predisposing to human disease. Here, we review the 3' end processing of the human F2 mRNA and demonstrate how clinically relevant mutations in the F2 gene contribute to thrombophilia by interfering with a tightly balanced architecture of noncanonical 3' end formation signals.
Generation of conditional alleles of the murine Iron Regulatory Protein (IRP)-1 and -2 genes.
Galy, B., Ferring, D. & Hentze, M.W.
Genesis. 2005 Dec;43(4):181-8.
Central aspects of cellular iron metabolism are controlled by IRP1 and IRP2, which are ubiquitously expressed in mouse organs and cells. Total and constitutive deficiency of both IRPs causes embryonic lethality in the mouse. To bypass the early lethality and to study organ-specific and/or temporal functions of IRP1 and/or IRP2 we generated Irp1 and Irp2 conditional alleles. We used mouse lines where a betaGeo gene trap construct was inserted into the second intron of the Irp1 and the Irp2 gene, generating hypomorphic alleles by interrupting the corresponding open reading frame near the amino-termini. The gene trap cassettes are flanked by Frt sites and were co-inserted with LoxP sites flanking exon 3. Flp-mediated removal of the gene trap construct generates floxed alleles with wildtype functions. For both Irp genes, Cre-assisted deletion of exon 3 generates complete null alleles that, in the case of IRP2, are associated with altered body iron distribution and compromised hematopoiesis. If not removed, the gene trap construct causes partially penetrant embryonic lethality unrelated to IRP deficiency when inserted within the Irp1 but not the Irp2 locus. We discuss the implications for functional genomics in the mouse.
Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements.
Gehring, N.H., Kunz, J.B., Neu-Yilik, G., Breit, S., Viegas, M.H., Hentze, M.W. & Kulozik, A.E.
Mol Cell 2005 Oct 7;20(1):65-75.
Messenger RNAs (mRNAs) bearing premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). For mammalian NMD, current models propose a linear pathway that involves the splicing-dependent deposition of exon-junction complexes (EJCs) and the sequential action of the NMD factors UPF3, UPF2, and UPF1. We show here that different EJC proteins serve as entry points for the formation of distinguishable NMD-activating mRNPs. Specifically, Y14, MAGOH, and eIF4A3 can activate NMD in an UPF2-independent manner, whereas RNPS1-induced NMD requires UPF2. We identify the relevant regions of RNPS1, eIF4A3, Y14, and MAGOH, which are essential for NMD and provide insights into the formation of complexes, that classify alternative NMD pathways. These results are integrated into a nonlinear model for mammalian NMD involving alternative routes of entry that converge at a common requirement of UPF1.
Altered body iron distribution and microcytosis in mice deficient in iron regulatory protein 2 (IRP2).
Galy, B., Ferring, D., Minana, B., Bell, O., Janser, H.G., Muckenthaler, M., Schumann, K. & Hentze, M.W.
Blood 2005 Oct 1;106(7):2580-9. Epub 2005 Jun 14.
Iron regulatory protein 2 (IRP2)-deficient mice have been reported to suffer from late-onset neurodegeneration by an unknown mechanism. We report that young adult Irp2-/- mice display signs of iron mismanagement within the central iron recycling pathway in the mammalian body, the liver-bone marrow-spleen axis, with altered body iron distribution and compromised hematopoiesis. In comparison with wild-type littermates, Irp2-/- mice are mildly microcytic with reduced serum hemoglobin levels and hematocrit. Serum iron and transferrin saturation are unchanged, and hence microcytosis is not due to an overt decrease in systemic iron availability. The liver and duodenum are iron loaded, while the spleen is iron deficient, associated with a reduced expression of the iron exporter ferroportin. A reduction in transferrin receptor 1 (TfR1) mRNA levels in the bone marrow of Irp2-/- mice can plausibly explain the microcytosis by an intrinsic defect in erythropoiesis due to a failure to adequately protect TfR1 mRNA against degradation. This study links a classic regulator of cellular iron metabolism to systemic iron homeostasis and erythropoietic TfR1 expression. Furthermore, this work uncovers aspects of mammalian iron metabolism that can or cannot be compensated for by the expression of IRP1.
Eukaryotic translation initiation factor 4GI and p97 promote cellular internal ribosome entry sequence-driven translation.
Hundsdoerfer, P., Thoma, C. & Hentze, M.W.
Proc Natl Acad Sci U S A 2005 Sep 20;102(38):13421-6. Epub 2005 Sep 7.
Numerous cellular mRNAs encoding proteins critical during cell stress, apoptosis, and the cell cycle seem to be translated by means of internal ribosome entry sequences (IRES) when cap-dependent translation is compromised. The underlying molecular mechanisms are largely unknown. Using a HeLa-based cell-free translation system that mirrors the function of cellular IRESs in vitro, we recently demonstrated that translation from the c-myc IRES continues after proteolytic cleavage of eukaryotic translation initiation factor (eIF) 4G. To address the role of eIF4G in cellular IRES-driven translation directly, we immunodepleted eIF4GI from the HeLa cell translation extracts. After efficient depletion of eIF4GI (>90%), both cap-dependent and c-myc IRES-dependent translations are diminished to residual levels (<5%). In striking contrast to cap-dependent translation, c-myc IRES-dependent translation is fully restored by addition of the conserved middle fragment of eIF4GI, harboring the eIF3- and eIF4A-binding sites. p97, an eIF4G-related protein that has been described both as an inhibitor of translation and as a modulator of apoptosis, not only suffices to also rescue c-myc IRES-driven (but not cap-dependent) translation, but it even superinduces IRES-mediated translation 3-fold compared with nondepleted extracts. Interestingly, both p97 and the middle fragment of eIF4GI also rescue translation driven by proapoptotic (p97) and antiapoptotic [X-linked inhibitor of apoptosis (XIAP) and cellular inhibitor of apoptosis 1 (c-IAP1)] IRESs, reflecting a broader role of these polypeptides in cellular IRES-mediated translation and indicating their importance in apoptosis.
A dual inhibitory mechanism restricts msl-2 mRNA translation for dosage compensation in Drosophila.
Beckmann, K., Grskovic, M., Gebauer, F. & Hentze, M.W.
Cell 2005 Aug 26;122(4):529-40.
Drosophila MSL-2 is the limiting component of the dosage compensation complex. Female flies must inhibit msl-2 mRNA translation for survival, and this inhibition is mediated by Sex-lethal (SXL) binding to sites in both the 5' and the 3' untranslated regions (UTRs). Here, we uncover the mechanism by which SXL achieves tight control of translation initiation. SXL binding to the 3'UTR regulatory region inhibits the recruitment of 43S ribosomal preinitiation complexes to the mRNA. Ribosomal complexes escaping this block and binding to the 5' end of the mRNA are challenged by SXL bound to the 5'UTR, which interferes with scanning to the downstream initiation codon of the mRNA. This failsafe mechanism thus forms the molecular basis of a critical step in dosage compensation. The results also elucidate a two step principle of translational control via multiple regulatory sites within an mRNA.
Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C Virus.
Fillebeen, C., Rivas-Estilla, A.M., Bisaillon, M., Ponka, P., Muckenthaler, M., Hentze, M.W., Koromilas, A.E. & Pantopoulos, K.
J Biol Chem 2005 Mar 11;280(10):9049-57. Epub 2005 Jan 6.
Clinical data suggest that iron is a negative factor in chronic hepatitis C; however, the molecular mechanisms by which iron modulates the infectious cycle of hepatitis C virus (HCV) remain elusive. To explore this, we utilized cells expressing a HCV replicon as a well-established model for viral replication. We demonstrate that iron administration dramatically inhibits the expression of viral proteins and RNA, without significantly affecting its translation or stability. Experiments with purified recombinant HCV RNA polymerase (NS5B) revealed that iron binds specifically and with high affinity (apparent Kd: 6 and 60 microM for Fe2+ and Fe3+, respectively) to the protein's Mg2+-binding pocket, thereby inhibiting its enzymatic activity. We propose that iron impairs HCV replication by inactivating NS5B and that its negative effects in chronic hepatitis C may be primarily due to attenuation of antiviral immune responses. Our data provide a direct molecular link between iron and HCV replication.
Molecular mechanisms of translational control.
Gebauer, F. & Hentze, M.W.
Nat Rev Mol Cell Biol 2004 Oct;5(10):827-35.
Translational control is widely used to regulate gene expression. This mode of regulation is especially relevant in situations where transcription is silent or when local control over protein accumulation is required. Although many examples of translational regulation have been described, only a few are beginning to be mechanistically understood. Instead of providing a comprehensive account of the examples that are known at present, we discuss instructive cases that serve as paradigms for different modes of translational control.
Enhancement of IRES-mediated translation of the c-myc and BiP mRNAs by the poly(A) tail is independent of intact eIF4G and PABP.
Thoma, C., Bergamini, G., Galy, B., Hundsdoerfer, P. & Hentze, M.W.
Mol Cell 2004 Sep 24;15(6):925-35.
The poly(A) tail at the 3' end of mRNAs enhances 5' cap-dependent translation initiation. We show that it also enhances IRES-directed translation of two cellular mRNAs in vitro and in vivo. The underlying mechanisms, however, differ fundamentally. In contrast to cap-dependent translation, IRES-driven translation continues to be enhanced by the poly(A) tail following proteolytic cleavage of eIF4G. Moreover, the poly(A) tail stimulates IRES-mediated translation even in the presence of PAIP2 or following effective depletion of the poly(A) binding protein (PABP) from HeLa cell extracts. The PABP-eIF4G bridging complex that is critical for cap-dependent translation is thus dispensable for the enhancement of the IRESs by the poly(A) tail. The polyadenylated mRNA translation from cellular IRESs is also profoundly sensitive to eIF4A activity in vitro. These mechanistic and molecular distinctions implicate the potential for a new layer of translational control mechanisms.
Molecular analysis of iron overload in beta2-microglobulin-deficient mice.
Muckenthaler, M.U., Rodrigues, P., Macedo, M.G., Minana, B., Brennan, K., Cardoso, E.M., Hentze, M.W. & de Sousa, M.
Blood Cells Mol Dis 2004 Sep-Oct;33(2):125-31.
Beta2-microglobulin knockout (beta2m-/-) mice represent an instructive model of spontaneous iron overload resembling genetic hemochromatosis. The mechanism of iron accumulation in this mouse model may be more complex than involving the MHC class I-like protein HFE. We report that beta2m-deficient mice, like Hfe-/- mice, lack the adaptive hepatic hepcidin mRNA increase to iron overload. The inverse correlation of hepatic iron levels and hepcidin mRNA expression in six beta2m-/- mice underlines the importance of hepcidin in regulating body iron stores. In contrast to Hfe-/- mice, beta2m-deficient mice display increased expression of the duodenal iron transporters DMT1 and ferroportin 1. This result implicates a broader role of beta2m in mammalian iron metabolism, suggesting that (an) additional beta2m-interacting protein(s) could be involved in controlling iron homeostasis, and highlighting the emerging connection of iron metabolism with the immune system.
Nonsense-mediated decay approaches the clinic.
Holbrook, J.A., Neu-Yilik, G., Hentze, M.W. & Kulozik, A.E.
Nat Genet 2004 Aug;36(8):801-8.
Nonsense-mediated decay (NMD) eliminates mRNAs containing premature termination codons and thus helps limit the synthesis of abnormal proteins. New results uncover a broader role of NMD as a pathway that also affects the expression of wild-type genes and alternative-splice products. Because the mechanisms by which NMD operates have received much attention, we discuss here the emerging awareness of the impact of NMD on the manifestation of human genetic diseases. We explore how an understanding of NMD accounts for phenotypic differences in diseases caused by premature termination codons. Specifically, we consider how the protective function of NMD sometimes benefits heterozygous carriers and, in contrast, sometimes contributes to a clinical picture of protein deficiency by inhibiting expression of partially functional proteins. Potential 'NMD therapeutics' will therefore need to strike a balance between the general physiological benefits of NMD and its detrimental effects in cases of specific genetic mutations.
The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations.
Danckwardt, S., Gehring, N.H., Neu-Yilik, G., Hundsdoerfer, P., Pforsich, M., Frede, U., Hentze, M.W. & Kulozik, A.E.
Blood 2004 Jul 15;104(2):428-35. Epub 2004 Apr 01.
The functional analysis of the common prothrombin 20210 G>A(F2 20210(*)A) mutation has recently revealed gain of function of 3'end processing as a novel genetic mechanism predisposing to human disease. We now show that the physiologic G at the cleavage site at position 20210 is the functionally least efficient nucleotide to support 3'end processing but has evolved to be physiologically optimal. Furthermore, the F2 3'end processing signal is characterized by a weak downstream cleavage stimulating factor (CstF) binding site with a low uridine density, and the functional efficiency of F2 3'end processing can be enhanced by the introduction of additional uridine residues. The recently identified thrombosis-related mutation (F2 20221(*)T) within the CstF binding site up-regulates F2 3'end processing and prothrombin biosynthesis in vivo. F2 20221(*)T thus represents the first example of a likely pathologically relevant mutation of the putative CstF binding site in the 3'flanking sequence of a human gene. Finally, we show that the low-efficiency F2 cleavage and CstF binding sites are balanced by a stimulatory upstream uridine-rich element in the 3'UTR. The architecture of the F2 3'end processing signal is thus characterized by a delicate balance of positive and negative signals. This balance appears to be highly susceptible to being disturbed by clinically relevant gain-of-function mutations.
Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo.
Galy, B., Ferring, D., Benesova, M., Benes, V. & Hentze, M.W.
RNA 2004 Jul;10(7):1019-25.
We report the targeted mutagenesis of the murine iron regulatory protein (IRP)-1 and IRP2 genes, respectively, with a classical gene trap construct. Insertion of the targeting cassette into the second intron of either gene by homologous recombination interrupts their open reading frames near the N termini. Mice that are homozygous for the correctly modified IRP1 or IRP2 alleles, respectively, display a strong reduction (90%, IRP1(-/-)) or nondetectable levels (IRP2(-/-)) of the targeted proteins. Interestingly, the pre-mRNAs transcribed from the identical targeting cassettes are processed differently within the two different contexts. Detailed analysis of the respective products identifies the choice of alternative splice and 3' end processing sites in the same tissues in vivo. We discuss the implications for the understanding of RNA processing and for targeting strategies for functional genomics in the mouse.
An Hfe-dependent pathway mediates hyposideremia in response to lipopolysaccharide-induced inflammation in mice.
Roy, C.N., Custodio, A.O., de Graaf, J., Schneider, S., Akpan, I., Montross, L.K., Sanchez, M., Gaudino, A., Hentze, M.W., Andrews, N.C. & Muckenthaler, M.U.
Nat Genet 2004 May;36(5):481-5. Epub 2004 Apr 18.
Inflammation influences iron balance in the whole organism. A common clinical manifestation of these changes is anemia of chronic disease (ACD; also called anemia of inflammation). Inflammation reduces duodenal iron absorption and increases macrophage iron retention, resulting in low serum iron concentrations (hyposideremia). Despite the protection hyposideremia provides against proliferating microorganisms, this 'iron withholding' reduces the iron available to maturing red blood cells and eventually contributes to the development of anemia. Hepcidin antimicrobial peptide (Hamp) is a hepatic defensin-like peptide hormone that inhibits duodenal iron absorption and macrophage iron release. Hamp is part of the type II acute phase response and is thought to have a crucial regulatory role in sequestering iron in the context of ACD. Mice with deficiencies in the hemochromatosis gene product, Hfe, mounted a general inflammatory response after injection of lipopolysaccharide but lacked appropriate Hamp expression and did not develop hyposideremia. These data suggest a previously unidentified role for Hfe in innate immunity and ACD.
Expression of epithelial cell iron-related genes upon infection by Neisseria meningitidis.
Bonnah, R.A., Muckenthaler, M.U., Carlson, H., Minana, B., Enns, C.A., Hentze, M.W. & So, M.
Cell Microbiol 2004 May;6(5):473-84.
Infection by the obligate human pathogens Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) reduces the expression of host epithelial cell transferrin receptor 1 (TfR-1) (Bonnah et al., 2000, Cellular Microbiology 2: 207-218). In addition, the rate and pattern of TfR-1 cycling is altered, leading to diminished uptake of Tf-iron by infected host cells. As Tf-iron is important for maintaining iron homeostasis in the eukaryotic cell, these findings raised the possibility that Neisseria infection might affect further pathways of epithelial cell iron metabolism. We used a specialized cDNA microarray platform, the 'IronChip', to investigate the expression of genes involved in iron transport, storage and regulation. We show that mRNA expression of several host genes involved in iron homeostasis is altered. Surprisingly, the general mRNA expression profile of infected cells closely resembled that of uninfected cells grown in an iron-limited environment. An important exception to this profile is TfR-1, the mRNA level of which is strongly reduced. Low TfR-1 expression may be explained in part by decreased activity of the iron-regulatory proteins (IRPs) in MC-infected cells, which may result in the destabilization of TfR-1 mRNA. Intriguingly, low IRP activity contrasts with the decrease in H-ferritin protein levels in infected cells. This finding suggests that low IRP activity may be responsible in part for the decrease in TfR-1 mRNA levels. A discussion of these novel findings in relation to MC infection and virulence is provided.
Balancing acts: molecular control of mammalian iron metabolism.
Hentze, M.W., Muckenthaler, M.U. & Andrews, N.C.
Cell 2004 Apr 30;117(3):285-97.
Iron is ubiquitous in the environment and in biology. The study of iron biology focuses on physiology and homeostasis-understanding how cells and organisms regulate their iron content, how diverse tissues orchestrate iron allocation, and how dysregulated iron homeostasis leads to common hematological, metabolic, and neurodegenerative diseases. This has provided novel insights into gene regulation and unveiled remarkable links to the immune system.
Rat duodenal IRP1 activity and iron absorption in iron deficiency and after HO perfusion.
Schumann, K., Brennan, K., Weiss, M., Pantopoulos, K. & Hentze, M.W.
Eur J Clin Invest 2004 Apr;34(4):275-82.
BACKGROUND: Iron regulatory protein 1 (IRP1), a post-transcriptional regulator of iron metabolism, is activated in the duodenum of iron-deficient animals, which is associated with increased iron absorption. In cell cultures IRP1 was also activated by iron-independent signals, such as H(2)O(2). Here we investigate whether luminal perfusion of rat duodenum with H(2)O(2) activates duodenal IRP1 and modulates duodenal iron absorption. METHODS: Duodena from iron-adequate Sprague-Dawley rats were luminally perfused with H(2)O(2). Iron regulatory protein-1 activity was determined in duodenal mucosa or in villus and crypt preparations by an electrophoretic mobility shift assay. Duodenal (59)Fe absorption was measured in isolated, perfused duodenal segments ex vivo and in ligated loops in vivo. (59)Fe uptake from the blood side was assessed after i.v. injection of (59)Fe-nitrilotriacetic acid. RESULTS: Similar to iron deficiency, the perfusion with 0-50 mM of H(2)O(2) increases duodenal IRP1 activity along the entire crypt villus-axis in a dose-dependent manner. After H(2)O(2) treatment, IRP1 remains activated for 12-24 h in the tips and for 72 h in the crypts. In iron-deficiency, IRP activation correlates with increased (59)Fe absorption. However, the H(2)O(2) treatment fails to stimulate any increase in (59)Fe uptake, without promoting damage of mucosal architecture or impairing glucose and water transport. CONCLUSION: Duodenal (59)Fe uptake is not affected by the H(2)O(2)-mediated activation of IRP1.
Iron-mediated degradation of IRP2, an unexpected pathway involving a 2-oxoglutarate-dependent oxygenase activity.
Wang, J., Chen, G., Muckenthaler, M., Galy, B., Hentze, M.W. & Pantopoulos, K.
Mol Cell Biol 2004 Feb;24(3):954-65.
Iron regulatory protein 2 (IRP2), a central posttranscriptional regulator of cellular and systemic iron metabolism, undergoes proteasomal degradation in iron-replete cells. The prevailing model postulates that the mechanism involves site-specific oxidation of 3 cysteine residues (C168, C174, and C178) within a 73-amino-acid (73-aa) degradation domain. By expressing wild-type and mutated versions of IRP2 in H1299 cells, we find that a C168S C174S C178S triple mutant, or a deletion mutant lacking the entire "73-aa domain," is sensitive to iron-mediated degradation, like wild-type IRP2. The antioxidants N-acetylcysteine, ascorbate, and alpha-tocopherol not only fail to stabilize IRP2 but, furthermore, promote its proteasomal degradation. The pathway for IRP2 degradation is saturable, which may explain earlier data supporting the "cysteine oxidation model," and shows remarkable similarities with the degradation of the hypoxia-inducible factor 1alpha (HIF-1alpha): dimethyl-oxalylglycine, a specific inhibitor of 2-oxoglutarate-dependent oxygenases, stabilizes IRP2 following the administration of iron to iron-deficient cells. Our results challenge the current model for IRP2 regulation and provide direct pharmacological evidence for the involvement of 2-oxoglutarate-dependent oxygenases in a pathway for IRP2 degradation.
Iron overload in adult Hfe-deficient mice independent of changes in the steady-state expression of the duodenal iron transporters DMT1 and Ireg1/ferroportin.
Herrmann, T., Muckenthaler, M., Van Der Hoeven, F., Brennan, K., Gehrke, S.G., Hubert, N., Sergi, C., Grone, H.J., Kaiser, I., Gosch, I., Volkmann, M., Riedel, H.D., Hentze, M.W., Stewart, A.F. & Stremmel, W.
J Mol Med 2004 Jan;82(1):39-48.
Patients suffering from hereditary hemochromatosis (HH) show progressive iron overload as a consequence of increased duodenal iron absorption. It has been hypothesized that mutations in the HH gene HFE cause misprogramming of the duodenal enterocytes towards a paradoxical iron-deficient state, resulting in increased iron transporter expression. Previous reports concerning gene expression levels of the duodenal iron transporters DMT1 and IREG1 in HH patients and animal models are controversial, however, and in many cases only mRNA expression levels were investigated. To analyze the duodenal expression of DMT1, Ireg1, Dcytb, and hephaestin and the association with iron overload in adult Hfe(-/-) mice, an Hfe(-/-) mouse line was generated. Duodenal DMT1 and Ireg1 protein levels, duodenal DMT1, Ireg1, Dcytb, hephaestin, and TfR1 mRNA levels, and hepatic hepcidin mRNA levels were quantified and the correlation to liver iron contents was calculated. We report that duodenal DMT1 and Ireg1 mRNA levels and DMT1 and Ireg1 protein levels remained unaffected by the Hfe deletion. Furthermore, duodenal hephaestin and TfR1 mRNA expression and hepatic hepcidin mRNA expression remained unaltered, while the duodenal mRNA expression of the brush border ferric reductase Dcytb was significantly increased in Hfe(-/-) mice. We found no correlation between the expression level of any of the analyzed transcripts and the liver iron content. In conclusion, the lack of correlation between DMT1 and Ireg1 protein expression and the liver iron content suggests that elevated duodenal iron transporter expression is not required for high liver iron overload. Hfe(-/-) mice do not necessarily display features of iron deficiency in the duodenum, indicated by an increase in mRNA and protein levels of DMT1 and Ireg1. Rather, the duodenal ferric reductase Dcytb may act as a possible mediator of iron overload in Hfe deficiency.
A poly(A) tail-responsive in vitro system for cap- or IRES-driven translation from HeLa cells.
Thoma, C., Ostareck-Lederer, A. & Hentze, M.W.
Methods Mol Biol. 2004;257:171-80.
In cells, the poly(A) tail stimulates translation from messenger RNAs bearing a cap structure or viral IRES elements. This 3' end-mediated stimulation of translation is not reflected in commonly used commercial cell-free translation systems prepared from rabbit reticulocytes or wheat germ. We describe a simple procedure to generate poly(A) tail-responsive translation extracts from HeLa cells. We suggest that this procedure should be adaptable to many animal cell lines.
Nonsense-mediated mRNA decay: from vacuum cleaner to Swiss army knife.
Neu-Yilik, G., Gehring, N.H., Hentze, M.W. & Kulozik, A.E.
Genome Biol 2004;5(4):218. Epub 2004 Mar 30.
Nonsense-mediated mRNA decay (NMD) downmodulates mRNAs that have in-frame premature termination codons and prevents translation of potentially harmful truncated proteins from aberrant mRNAs. Two new approaches have identified physiological NMD substrates, and suggest that NMD functions as a multipurpose tool in the modulation of gene expression.
Homodirectional changes in transcriptome composition and mRNA translation induced by rapamycin and heat shock.
Preiss, T., Baron-Benhamou, J., Ansorge, W. & Hentze, M.W.
Nat Struct Biol 2003 Dec;10(12):1039-47. Epub 2003 Nov 9.
Transcription and mRNA turnover determine the quantitative composition of the cellular transcriptome. The transcriptome in turn serves as a template for the proteome via translation. Treatment of Saccharomyces cerevisiae with the TOR kinase inhibitor rapamycin causes increases and decreases in the mRNA levels of hundreds of genes. We used DNA microarray analysis to monitor simultaneously transcriptome and translational changes for all detectable yeast mRNAs. Notably, genes that are induced in the transcriptome correlate tightly with more efficiently translated mRNAs (based on their relative degree of polyribosome association); similarly, genes that show reduced mRNA levels after rapamycin treatment also show lower translational fitness. Microarray analyses on heat-shocked cells also reveal homodirectional co-regulatory responses. Thus, signal-induced changes in the transcriptome are amplified at the translational level. These results unveil a higher level of coordinated gene regulation that we refer to as 'potentiation.'
Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7.
Ohnishi, T., Yamashita, A., Kashima, I., Schell, T., Anders, K.R., Grimson, A., Hachiya, T., Hentze, M.W., Anderson, P. & Ohno, S.
Mol Cell 2003 Nov;12(5):1187-200.
Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.
A co-repressor assembly nucleated by Sex-lethal in the 3'UTR mediates translational control of Drosophila msl-2 mRNA.
Grskovic, M., Hentze, M.W. & Gebauer, F.
EMBO J 2003 Oct 15;22(20):5571-81.
Drosophila Sex-lethal (dSXL)-mediated translational repression of male-specific lethal 2 (msl-2) mRNA is essential for X-chromosome dosage compensation. Binding of dSXL to specific sites in both untranslated regions of msl-2 mRNA is necessary for inhibition of translation initiation. We describe the organization of dSXL as a translational regulator and show that the RNA binding and translational repressor functions are contained within the two RRM domains and a C-terminal heptapeptide extension. The repressor function is dormant unless dSXL binds to msl-2 mRNA with its own RRMs, because dSXL tethering via a heterologous RNA-binding peptide does not elicit translational inhibition. We reveal proteins that crosslink to the msl-2 3' untranslated region (3'UTR) and co-immunoprecipitate with dSXL in a fashion that requires its intact repressor domain and correlates with translational regulation. Translation competition and UV-crosslink experiments show that the 3'UTR msl-2 sequences adjacent to dSXL-binding sites are necessary to recruit titratable co-repressors. Our data support a model where dSXL binding to the 3'UTR of msl-2 mRNA activates the translational repressor domain, thereby enabling it to recruit co-repressors in a specific fashion.
Complexes between the nonsense-mediated mRNA decay pathway factor human upf1 (up-frameshift protein 1) and essential nonsense-mediated mRNA decay factors in HeLa cells.
Schell, T., Kocher, T., Wilm, M., Seraphin, B., Kulozik, A.E. & Hentze, M.W.
Biochem J 2003 Aug 1;373(Pt 3):775-83.
mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals. To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generated a HeLa cell line that stably expresses Hupf1 bearing a double-affinity tag (termed Hupf1-2tag). Hupf1-2tag is localized in the cytoplasm similar to the endogenous Hupf1 protein, and the Hupf1-2tag cell line is fully NMD-competent. Using affinity chromatography, Hupf1-2tag-associated proteins were isolated. MS and immunoblotting identified the NMD factors Hupf2 and Hupf3a/b as interaction partners of Hupf1. Size-exclusion chromatography indicates that the NMD factors Hupf1, Hupf2 and the large isoform of Hupf3a might exist in a stable, high-molecular-mass complex of approx. 1.3 MDa. Interestingly, the poly(A)-binding protein was also identified by MS to be associated specifically with Hupf1-2tag. In contrast with the interaction with Hupf2 and Hupf3a/b, the association of poly(A)-binding protein with Hupf1 is highly sensitive to treatment of the isolated complexes with RNase. Components of the exon-exon junction complex or the translational eukaryotic release factor (eRF) 3 were not identified in complexes associated with Hupf1-2tag. We discuss these findings in the context of current models of NMD.
The interaction of the cap-binding complex (CBC) with eIF4G is dispensable for translation in yeast.
Baron-Benhamou, J., Fortes, P., Inada, T., Preiss, T. & Hentze, M.W.
RNA 2003 Jun;9(6):654-62.
In eukaryotes, the m(7)GpppN cap structure is added to all nascent RNA polymerase II transcripts, and serves important functions at multiple steps of RNA metabolism. The predominantly nuclear cap-binding complex (CBC) binds to the cap during RNA synthesis. The predominantly cytoplasmic eukaryotic initiation factor 4F (eIF4F) is thought to replace CBC after export of mature mRNA to the cytoplasm, and mediates the bulk of cellular translation. Yeast as well as mammalian CBC interacts in vitro with eIF4G, a subunit of eIF4F. In this work, we investigate a potential role of this interaction during translation in yeast. We identify a mutation (DR548/9AA) in Tif4631p, one of two isoforms of yeast eIF4G, that abolishes its binding to CBC. Cells expressing this mutant protein as the sole source of eIF4G grow at wild-type rates, and bulk cellular translation, as assessed by metabolic labeling and polysome profile analysis, is unchanged. Importantly, we find that the DR548/9AA mutation neither diminishes nor delays the translation of newly induced reporter mRNA. Finally, microarray analysis reveals marked transcriptome alterations in CBC subunit deletion strains, whereas eIF4G point mutants have essentially a wild-type transcriptome composition. Collectively, these data suggest that in yeast, the phenotypic consequences of CBC deletions are separable from its interaction with eIF4G, and that the CBC-eIF4G interaction is dispensable for a potential "pioneering round" of translation in yeast.
Relationships and distinctions in iron-regulatory networks responding to interrelated signals.
Muckenthaler, M., Richter, A., Gunkel, N., Riedel, D., Polycarpou-Schwarz, M., Hentze, S., Falkenhahn, M., Stremmel, W., Ansorge, W. & Hentze, M.W.
Blood 2003 May 1;101(9):3690-8. Epub 2002 Sep 26.
Specialized cDNA-based microarrays (IronChips) were developed to investigate complex physiological gene-regulatory patterns in iron metabolism. Approximately 115 human cDNAs were strategically selected to represent genes involved either in iron metabolism or in interlinked pathways (eg, oxidative stress, nitric oxide [NO] metabolism, or copper metabolism), and were immobilized on glass slides. HeLa cells were treated with iron donors or iron chelators, or were subjected to oxidative stress (H(2)O(2)) or NO (sodium nitroprusside). In addition, we generated a stable transgenic HeLa cell line expressing the HFE gene under an inducible promoter. Gene-response patterns were recorded for all of these interrelated experimental stimuli, and analyzed for common and distinct responses that define signal-specific regulatory patterns. The resulting regulatory patterns reveal and define degrees of relationship between distinct signals. Remarkably, the gene responses elicited by the altered expression of the hemochromatosis protein HFE and by pharmacological iron chelation exhibit the highest degree of relatedness, both for iron-regulatory protein (IRP) and non-IRP target genes. This finding suggests that HFE expression directly affects the intracellular chelatable iron pool in the transgenic cell line. Furthermore, cells treated with the iron donors hemin or ferric ammonium citrate display response patterns that permit the identification of the iron-loaded state in both cases, and the discrimination between the sources of iron loading. These findings also demonstrate the broad utility of gene-expression profiling with the IronChip to study iron metabolism and related human diseases.
Regulatory defects in liver and intestine implicate abnormal hepcidin and Cybrd1 expression in mouse hemochromatosis.
Muckenthaler, M., Roy, C.N., Custodio, A.O., Minana, B., deGraaf, J., Montross, L.K., Andrews, N.C. & Hentze, M.W.
Nat Genet 2003 May;34(1):102-7.
Individuals with hereditary hemochromatosis suffer from systemic iron overload due to duodenal hyperabsorption. Most cases arise from a founder mutation in HFE (845G-->A; ref. 2) that results in the amino-acid substitution C282Y and prevents the association of HFE with beta2-microglobulin. Mice homozygous with respect to a null allele of Hfe (Hfe-/-) or homozygous with respect to the orthologous 882G-->A mutation (Hfe(845A/845A)) develop iron overload that recapitulates hereditary hemochromatosis in humans, confirming that hereditary hemochromatosis arises from loss of HFE function. Much work has focused on an exclusive role for the intestine in hereditary hemochromatosis. HFE deficiency in intestinal crypt cells is thought to cause intestinal iron deficiency and greater expression of iron transporters such as SLC11A2 (also called DMT1, DCT1 and NRAMP2) and SLC11A3 (also called IREG1, ferroportin and MTP1; ref. 3). Published data on the expression of these transporters in the duodenum of HFE-deficient mice and humans are contradictory. In this report, we used a custom microarray to assay changes in duodenal and hepatic gene expression in Hfe-deficient mice. We found unexpected alterations in the expression of Slc39a1 (mouse ortholog of SLC11A3) and Cybrd1, which encode key iron transport proteins, and Hamp (hepcidin antimicrobial peptide), a hepatic regulator of iron transport. We propose that inappropriate regulatory cues from the liver underlie greater duodenal iron absorption, possibly involving the ferric reductase Cybrd1.
Drosophila sex-lethal inhibits the stable association of the 40S ribosomal subunit with msl-2 mRNA.
Gebauer, F., Grskovic, M. & Hentze, M.W.
Mol Cell 2003 May;11(5):1397-404.
The inhibition of male-specific lethal-2 (msl-2) mRNA translation in female flies is essential for X chromosome dosage compensation in Drosophila melanogaster. Translational repression of msl-2 requires sex-lethal (SXL) binding to uridine-rich sequences in both the 5' and 3' untranslated regions (UTRs) of the message. We delineate the msl-2 mRNA sequence elements that are important for regulation by SXL and identify functionally critical sequences adjacent to regulatory SXL binding sites. We demonstrate that SXL inhibits translation initiation and prevents the stable association of the 40S ribosomal subunit with the mRNA in a manner that does not require the presence of a cap structure at the 5' end of the mRNA. These results elucidate a critical regulatory step for dosage compensation in Drosophila melanogaster.
Y14 and hUpf3b form an NMD-activating complex.
Gehring, N.H., Neu-Yilik, G., Schell, T., Hentze, M.W. & Kulozik, A.E.
Mol Cell 2003 Apr;11(4):939-49.
Messenger RNAs with premature translation termination codons (PTCs) are degraded by nonsense-mediated mRNA decay (NMD). In mammals, PTCs are discriminated from physiological stop codons by a process thought to involve the splicing-dependent deposition of an exon junction complex (EJC), EJC-mediated recruitment of Upf3, and Upf2 binding to the N terminus of Upf3. Here, we identify a conserved domain of hUpf3b that mediates an interaction with the EJC protein Y14. Tethered function analysis shows that the Y14/hUpf3b interaction is essential for NMD, while surprisingly the interaction between hUpf3b and hUpf2 is not. Nonetheless, hUpf2 is necessary for NMD mediated by tethered Y14. RNAi-induced knockdown and Y14 repletion of siRNA-treated cells implicates Y14 in the degradation of beta-globin NS39 mRNA and demonstrates that Y14 is required for NMD induced by tethered hUpf3b. These results uncover a direct role of Y14 in NMD and suggest an unexpected hierarchy in the assembly of NMD complexes.
Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress.
Thompson, K., Menzies, S., Muckenthaler, M., Torti, F.M., Wood, T., Torti, S.V., Hentze, M.W., Beard, J. & Connor, J.
J Neurosci Res 2003 Jan 1;71(1):46-63.
Several neurodegenerative disorders such as Parkinson's Disease (PD) and Alzheimer's Disease (AD) are associated with elevated brain iron accumulation relative to the amount of ferritin, the intracellular iron storage protein. The accumulation of more iron than can be adequately stored in ferritin creates an environment of oxidative stress. We developed a heavy chain (H) ferritin null mutant in an attempt to mimic the iron milieu of the brain in AD and PD. Animals homozygous for the mutation die in utero but the heterozygotes (+/-) are viable. We examined heterozygous and wild-type (wt) mice between 6 and 8 months of age. Macroscopically, the brains of +/- mice were well formed and did not differ from control brains. There was no evidence of histopathology in the brains of the heterozygous mice. Iron levels in the brain of the +/- and wild-type (+/+) mice were similar, but +/- mice had less than half the levels of H-ferritin. The other iron management proteins transferrin, transferrin receptor, light chain ferritin, Divalent Metal Transporter 1, ceruloplasmin, were increased in the +/- mice compared to +/+ mice. The relative amounts of these proteins in relation to the iron concentration are similar to that found in AD and PD. Thus, we hypothesized that the brains of the heterozygote mice should have an increase in indices of oxidative stress. In support of this hypothesis, there was a decrease in total superoxide dismutase (SOD) activity in the heterozygotes coupled with an increase in oxidatively modified proteins. In addition, apoptotic markers Bax and caspase-3 were detected in neurons of the +/- mice but not in the wt. Thus, we have developed a mouse model that mimics the protein profile for iron management seen in AD and PD that also shows evidence of oxidative stress. These results suggest that this mouse may be a model to determine the role of iron mismanagement in neurodegenerative disorders and for testing antioxidant therapeutic strategies.
Previously uncharacterized isoforms of divalent metal transporter (DMT)-1: implications for regulation and cellular function.
Hubert, N. & Hentze, M.W.
Proc Natl Acad Sci U S A 2002 Sep 17;99(19):12345-50.
Divalent metal transporter 1 (DMT1) mediates apical iron uptake into duodenal enterocytes and also transfers iron from the endosome into the cytosol after cellular uptake via the transferrin receptor. Hence, mutations in DMT1 cause systemic iron deficiency and anemia. DMT1 mRNA levels are increased in the duodenum of iron-deficient animals. This regulation has been observed for DMT1 mRNA harboring an iron-responsive element (IRE) in its 3' UTR, but not for a processing variant lacking a 3'UTR IRE, suggesting that the IRE regulates the expression of DMT1 mRNA in response to iron levels. Here, we show that iron regulation of DMT1 involves the expression of a previously unrecognized upstream 5' exon (exon 1A) of the human and murine DMT1 gene. The expression of this previously uncharacterized 5' exon is tissue-specific and particularly prevalent in the duodenum and kidney. It adds an in-frame AUG translation initiation codon extending the DMT1 ORF by a conserved sequence of 29-31 amino acids. In combination with the IRE- and non-IRE variants in the 3'UTR, our results reveal the existence of four DMT1 mRNA isoforms predicting the synthesis of four different DMT1 proteins. We show that two regulatory regions, the 5' promoter/exon 1A region and the IRE-containing terminal exon participate in iron regulation of DMT1 expression, which operate in a tissue-specific way. These results uncover an unexpected complexity of DMT1 expression and regulation, with implications for understanding the physiology, cell biology, and pathophysiology of mammalian iron metabolism.
Tolerable upper intakes for dietary iron set by the US Food and Nutrition Board.
Schumann, K., Borch-Iohnsen, B., Hentze, M.W. & Marx, J.J.
Am J Clin Nutr 2002 Sep;76(3):499-500. Europe PMC
Comparison of fluorescent tag DNA labeling methods used for expression analysis by DNA microarrays.
Richter, A., Schwager, C., Hentze, S., Ansorge, W., Hentze, M.W. & Muckenthaler, M.
Biotechniques 2002 Sep;33(3):620-8, 630.
Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.
c-Src-mediated phosphorylation of hnRNP K drives translational activation of specifically silenced mRNAs.
Ostareck-Lederer, A., Ostareck, D.H., Cans, C., Neubauer, G., Bomsztyk, K., Superti-Furga, G. & Hentze, M.W.
Mol Cell Biol 2002 Jul;22(13):4535-43.
hnRNPK and hnRNP E1/E2 mediate translational silencing of cellular and viral mRNAs in a differentiation-dependent way by binding to specific regulatory sequences. The translation of 15-lipoxygenase (LOX) mRNA in erythroid precursor cells and of the L2 mRNA of human papilloma virus type 16 (HPV-16) in squamous epithelial cells is silenced when either of these cells is immature and is activated in maturing cells by unknown mechanisms. Here we address the question of how the silenced mRNA can be translationally activated. We show that hnRNP K and the c-Src kinase specifically interact with each other, leading to c-Src activation and tyrosine phosphorylation of hnRNP K in vivo and in vitro. c-Src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3' untranslated region in vitro and specifically derepresses the translation of DICE-bearing mRNAs in vivo. Our results establish a novel role of c-Src kinase in translational gene regulation and reveal a mechanism by which silenced mRNAs can be translationally activated.
Abnormally spliced beta-globin mRNAs: a single point mutation generates transcripts sensitive and insensitive to nonsense-mediated mRNA decay.
Danckwardt, S., Neu-Yilik, G., Thermann, R., Frede, U., Hentze, M.W. & Kulozik, A.E.
Blood 2002 Mar 1;99(5):1811-6.
Nonsense-mediated mRNA decay (NMD) represents a phylogenetically widely conserved splicing- and translation-dependent mechanism that eliminates transcripts with premature translation stop codons and suppresses the accumulation of C-terminally truncated peptides. Elimination of frameshifted transcripts that result from faulty splicing may be an important function of NMD. To test this hypothesis directly, this study used the IVS1 + 5 G>A thalassemia mutation of the human beta-globin gene as a model system. We generated beta-globin gene constructs with this mutation and an iron-responsive element in the 5' untranslated region, which allowed specific experimental activation and inactivation of translation and, hence, NMD of this transcript. Premessenger RNAs with IVS1 + 5 G>A were spliced at normal sites and cryptic sites, enabling a direct comparison of the effect of NMD on the accumulation of normal and frameshifted messenger RNAs. In transfected HeLa cells, the predominant frameshifted transcript was degraded under conditions of active NMD, whereas accumulation to high levels occurred under conditions of specifically disabled NMD, thereby indicating an important physiologic function of NMD in the control of the splicing process. An unexpected finding was that accumulation of a second aberrant transcript remained unaffected by NMD. The IVS1 + 5 G>A mutation thus revealed the presence of an unknown cis-acting determinant that influences the NMD sensitivity of a putative NMD substrate. It can therefore serve as a useful tool for defining the mechanisms that permit specific transcripts to circumvent the NMD pathway.
The human intronless melanocortin 4-receptor gene is NMD insensitive.
Brocke, K.S., Neu-Yilik, G., Gehring, N.H., Hentze, M.W. & Kulozik, A.E.
Hum Mol Genet 2002 Feb 1;11(3):331-335.
Nonsense-mediated decay (NMD) is a phylogenetically widely conserved mechanism that contributes to the fidelity of gene expression. NMD inhibits the accumulation of nonsense- or frameshift-mutated mRNA and thus minimizes the synthesis of truncated proteins with potential dominant negative effects. Yeast and higher eukaryotes use somewhat diverse mechanisms to promote NMD and to discriminate between premature and physiological translation termination codons. NMD in yeast involves the binding of specific RNA-binding proteins to cis-acting exonic elements. In contrast, NMD of the intron-containing genes of higher eukaryotes is splicing-dependent. Here, we investigated the NMD sensitivity of nonsense-mutated transcripts of the naturally intronless human melanocortin 4-receptor (MC4-R) gene. Nonsense-mutated variants of MC4-R transcripts are stable and express truncated proteins that are detectable in the lysates of transfected cells. Thus, the naturally intronless MC4-R gene and probably many other intronless genes fail to be monitored by the NMD pathway.
Integration of splicing, transport and translation to achieve mRNA quality control by the nonsense-mediated decay pathway.
Schell, T., Kulozik, A.E. & Hentze, M.W.
Genome Biol 2002;3(3):REVIEWS1006.
When pre-mRNAs are spliced, a multi-component complex is deposited onto them, close to the sites of intron removal. New findings suggest that these exon-exon junction complexes and the complexes that bind mRNA caps are key effectors of the fate of spliced mRNAs and may regulate whether mRNAs containing premature stop codons are degraded.
Iron-dependent regulation of the divalent metal ion transporter.
Gunshin, H., Allerson, C.R., Polycarpou-Schwarz, M., Rofts, A., Rogers, J.T., Kishi, F., Hentze, M.W., Rouault, T.A., Andrews, N.C. & Hediger, M.A.
FEBS Lett 2001 Dec 7;509(2):309-16.
The first step in intestinal iron absorption is mediated by the H(+)-coupled Fe(2+) transporter called divalent cation transporter 1/divalent metal ion transporter 1 (DCT1/DMT1) (also known as natural resistance-associated macrophage protein 2). DCT1/DMT1 mRNA levels in the duodenum strongly increase in response to iron depletion. To study the mechanism of iron-dependent DCT1/DMT1 mRNA regulation, we investigated the endogenous expression of DCT1/DMT1 mRNA in various cell types. We found that only the iron responsive element (IRE)-containing form, which corresponds to one of two splice forms of DCT1/DMT1, is responsive to iron treatment and this responsiveness was cell type specific. We also examined the interaction of the putative 3'-UTR IRE with iron responsive binding proteins (IRP1 and IRP2), and found that IRP1 binds to the DCT1/DMT1-IRE with higher affinity compared to IRP2. This differential binding of IRP1 and IRP2 was also reported for the IREs of transferrin receptors, erythroid 5-aminolevulinate synthase and mitochondrial aconitase. We propose that regulation of DCT1/DMT1 mRNA by iron involves post-transcriptional regulation through the binding of IRP1 to the transporter's IRE, as well as other as yet unknown factors.
Generation of stable mRNA fragments and translation of N-truncated proteins induced by antisense oligodeoxynucleotides.
Thoma, C., Hasselblatt, P., Kock, J., Chang, S.F., Hockenjos, B., Will, H., Hentze, M.W., Blum, H.E., von Weizsacker, F. & Offensperger, W.B.
Mol Cell 2001 Oct;8(4):865-72.
Binding of phosphorothioate-modified antisense oligodeoxynucleotides (AS ODNs) to target mRNAs is commonly thought to mediate RNA degradation or block of translation. Here we demonstrate cleavage of target mRNAs within the AS ODN binding region with subsequent degradation of the 5' but not the 3' cleavage product. Some, if not all, 3' mRNA fragments lacked a 5' cap structure, whereas their poly(A) tail length remained unchanged. Furthermore, they were efficiently translated into N-terminally truncated proteins as demonstrated in three settings: production of shortened hepadnaviral surface proteins, alteration of the subcellular localization of a fluorescent protein, and shift of the transcription factor C/EBPalpha isoform expression levels. Thus, AS treatment may result in the synthesis of N-truncated proteins with biologically relevant effects.
Protein synthesis. Believe it or not-translation in the nucleus.
Science. 2001 Aug 10;293(5532):1058-9. Europe PMC
Increased efficiency of mRNA 3' end formation: a new genetic mechanism contributing to hereditary thrombophilia.
Gehring, N.H., Frede, U., Neu-Yilik, G., Hundsdoerfer, P., Vetter, B., Hentze, M.W. & Kulozik, A.E.
Nat Genet 2001 Aug;28(4):389-92.
The G-->A mutation at position 20210 of the prothrombin or coagulation factor II gene (F2) represents a common genetic risk factor for the occurrence of thromboembolic events. This mutation affects the 3'-terminal nucleotide of the 3' untranslated region (UTR) of the mRNA and causes elevated prothrombin plasma concentrations by an unknown mechanism. Here, we show that the mutation does not affect the amount of pre-mRNA, the site of 3' end cleavage or the length of the poly(A) tail of the mature mRNA. Rather, we demonstrate that the physiological F2 3' end cleavage signal is inefficient and that F2 20210 G-->A represents a gain-of-function mutation, causing increased cleavage site recognition, increased 3' end processing and increased mRNA accumulation and protein synthesis. Enhanced mRNA 3' end formation efficiency emerges as a novel principle causing a genetic disorder and explains the role of the F2 20210 G-->A mutation in the pathogenesis of thrombophilia. This work also illustrates the pathophysiologic importance of quantitatively minor aberrations of RNA metabolism.
Fertility facts: male and female germ cell development requires translational control by CPEB.
Gebauer, F. & Hentze, M.W.
Mol Cell 2001 Aug;8(2):247-9.
In the August issue of Developmental Cell, Tay and Richter examine the consequences of eliminating CPEB function in mice. Their studies reveal an important role for this translational regulator at the pachytene stage of germ cell differentiation.
IRP1 activation by extracellular oxidative stress in the perfused rat liver.
Mueller, S., Pantopoulos, K., Hubner, C.A., Stremmel, W. & Hentze, M.W.
J Biol Chem 2001 Jun 22;276(25):23192-6.
The expression of several proteins with critical functions in iron metabolism is regulated post-transcriptionally by the binding of iron regulatory proteins, IRP1 and IRP2, to mRNA iron responsive elements (IREs). In iron-deficient tissues and cultured cells, both IRP1 and IRP2 are activated for high affinity IRE binding. Previous work showed that IRP1 is also activated when cultured cells are exposed to H(2)O(2). The well established role of iron and H(2)O(2) in tissue injury (based on Fenton chemistry) suggests that this response may have important pathophysiological implications. This is particularly relevant in inflammation, where cytotoxic immune cells release large amounts of reactive oxygen species. Here, we describe a rat liver perfusion model to study IRP1 activation under H(2)O(2) generation conditions that mimic a physiological inflammatory response, using steady-state concentrations of H(2)O(2) produced by a glucose/ glucose oxidase/catalase system. We show first that stimulated neutrophils are able to increase serum levels of H(2)O(2) by a factor of 10, even in the presence of H(2)O(2)-degrading erythrocytes. We further show that perfusion of rat liver with glucose oxidase leads to a rapid activation of IRE binding activity in the intact organ. Mobility shift assays with liver extracts and IRP1 or IRP2-specific probes indicate that only IRP1 responds to H(2)O(2). Our study demonstrates a principal existence of iron regulation by oxidative stress at the intact organ level. It also provides a link between iron metabolism and the inflammatory response, as H(2)O(2) is a major product of the oxidative burst of neutrophils and macrophages.
Construction of regulatable picornavirus IRESes as a test of current models of the mechanism of internal translation initiation.
Poyry, T.A., Hentze, M.W. & Jackson, R.J.
RNA 2001 May;7(5):647-60.
Picornavirus internal ribosome entry sites (IRESs) are approximately 450 nt. RNA elements that direct internal initiation of translation, such that when placed between the two cistrons of a dicistronic construct, they drive independent translation of the downstream cistron. Consequently they have been widely used for coordinated expression of two or more proteins. All picornavirus IRESs have an AUG triplet at the very 3' end, which is thought to be the actual site of internal ribosome entry. However with some IRESs, such as foot-and-mouth disease virus, and especially poliovirus, the majority of ribosomes do not initiate translation at this putative entry site AUG, but at the next AUG further downstream, which is thought to be accessed by a process of linear ribosome scanning from the entry site. If this is so, then it should be possible to regulate IRES-dependent translation by inserting an iron responsive element (IRE) between the putative entry site AUG and the main functional initiation site. This should make IRES-dependent translation sensitive to the concentration of iron regulatory protein (IRP), the protein that specifically binds to the IRE. This has been attempted with both the foot-and-mouth disease virus and poliovirus IRESs, and was successful in so far as an inhibition specifically of IRES-dependent translation was observed that was strictly dependent on both the presence of IRP and of a functional IRE motif inserted in the sense orientation. However, the range over which expression could be varied was rather limited (three- to fourfold maximum), because some IRES-dependent translation remained completely refractory to inhibition by even very high IRP concentrations. In contrast, with a cap-proximal IRE in the 5' untranslated region of an mRNA translated by the scanning mechanism, addition of sufficient IRP results in complete inhibition. These results support the model of IRES-promoted ribosome entry at an upstream site followed by strictly linear scanning to the main functional initiation site for the majority of internal initiation events, but imply that some ribosomes must access the functional initiation site by another route, possibly a nonlinear shunting-like mechanism.
ERK phosphorylation drives cytoplasmic accumulation of hnRNP-K and inhibition of mRNA translation.
Habelhah, H., Shah, K., Huang, L., Ostareck-Lederer, A., Burlingame, A.L., Shokat, K.M., Hentze, M.W. & Ronai, Z.
Nat Cell Biol 2001 Mar;3(3):325-30.
Heterogeneous nuclear ribonucleoprotein K (hnRNP-K) is one of a family of 20 proteins that are involved in transcription and post-transcriptional messenger RNA metabolism. The mechanisms that underlie regulation of hnRNP-K activities remain largely unknown. Here we show that cytoplasmic accumulation of hnRNP-K is phosphorylation-dependent. Mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) efficiently phosphorylates hnRNP-K both in vitro and in vivo at serines 284 and 353. Serum stimulation or constitutive activation of ERK kinase (MEK1) results in phosphorylation and cytoplasmic accumulation of hnRNP-K. Mutation at ERK phosphoacceptor sites in hnRNP-K abolishes the ability to accumulate in the cytoplasm and renders the protein incapable of regulating translation of mRNAs that have a differentiation-control element (DICE) in the 3' untranslated region (UTR). Similarly, treatment with a pharmacological inhibitor of the ERK pathway abolishes cytoplasmic accumulation of hnRNP-K and attenuates inhibition of mRNA translation. Our results establish the role of MAPK/ERK in phosphorylation-dependent cellular localization of hnRNP-K, which is required for its ability to silence mRNA translation.
Splicing and 3' end formation in the definition of nonsense-mediated decay-competent human beta-globin mRNPs.
Neu-Yilik, G., Gehring, N.H., Thermann, R., Frede, U., Hentze, M.W. & Kulozik, A.E.
EMBO J 2001 Feb 1;20(3):532-40.
Premature translation termination codons are common causes of genetic disorders. mRNAs with such mutations are degraded by a surveillance mechanism termed nonsense-mediated decay (NMD), which represents a phylogenetically widely conserved post-transcriptional mechanism for the quality control of gene expression. How NMD-competent mRNPs are formed and specified remains a central question. Here, we have used human beta-globin mRNA as a model system to address the role of splicing and polyadenylation for human NMD. We show that (i) splicing is an indispensable component of the human beta-globin NMD pathway, which cannot be compensated for by exonic beta-globin 'failsafe' sequences; (ii) the spatial requirements of human beta-globin NMD, as signified by the maximal distance of the nonsense mutation to the final exon-exon junction, are less constrained than in yeast; and (iii) non-polyadenylated mRNAs with a histone 3' end are NMD competent. Thus, the formation of NMD-competent mRNP particles critically depends on splicing but does not require the presence of a poly(A) tail.
Lipoxygenase mRNA silencing in erythroid differentiation: The 3'UTR regulatory complex controls 60S ribosomal subunit joining.
Ostareck, D.H., Ostareck-Lederer, A., Shatsky, I.N. & Hentze, M.W.
Cell 2001 Jan 26;104(2):281-90.
15-lipoxygenase (LOX) expression is translationally silenced in early erythroid precursor cells by a specific mRNA-protein complex formed between the differentiation control element in the 3' untranslated region (UTR) and hnRNPs K and E1. The 3'UTR regulatory complex prevents translation initiation by an unknown mechanism. We demonstrate that the 40S ribosomal subunit can be recruited and scan to the translation initiation codon even when the silencing complex is bound to the 3'UTR. However, the joining of the 60S ribosomal subunit at the AUG codon to form a translation competent 80S ribosome is inhibited, unless initiation is mediated by the IGR-IRES of the cricket paralysis virus. These findings identify the critical step at which LOX mRNA translation is controlled and reveal that 60S subunit joining can be specifically regulated.
Tethered-function analysis reveals that elF4E can recruit ribosomes independent of its binding to the cap structure.
De Gregorio, E., Baron, J., Preiss, T. & Hentze, M.W.
RNA 2001 Jan;7(1):106-13.
The cap-binding complex elF4F is involved in ribosome recruitment during the initiation phase of translation and is composed of three subunits: elF4E, -4G, and -4A. The m7GpppN cap-binding subunit eIF4E binds the N-terminal region of eIF4G, which in turn contacts eIF4A through its central and C-terminal regions. We have previously shown, through a tethered-function approach in transfected HeLa cells, that the binding of eIF4G to an mRNA is sufficient to drive productive translation (De Gregorio et al., EMBO J, 1999, 18:4865-4874). Here we exploit this approach to assess which of the other subunits of elF4F can exert this function. eIF4AI or mutant forms of eIF4E were fused to the RNA-binding domain of the lambda phage antiterminator protein N to generate the chimeric proteins lambda4A, lambda4E-102 (abolished cap binding), and lambda4E-73-102 (impaired binding to both, the cap and eIF4G). The fusion proteins were directed to a bicistronic reporter mRNA by means of interaction with a specific lambda-N binding site (boxB) in the intercistronic space. We show that lambda4E-102, but neither the double mutant lambda4E-73-102 nor lambda4A, suffices to promote translation of the downstream gene in this assay. Coimmunoprecipitation analyses confirmed that all lambda-fusion proteins are capable of interacting with the appropriate endogenous eIF4F subunits. These results reveal that eIF4E, as well as eIF4G, can drive ribosome recruitment independent of a physical link to the cap structure. In spite of its interaction with endogenous eIF4G, lambda4A does not display this property. eIF4A thus appears to supply an essential auxiliary function to eIF4F that may require its ability to cycle into and out of this complex.
Translational control of 15-lipoxygenase and msl-2 mRNAs: single regulators or corepressor assemblies?
Gebauer, F., Ostareck, D.H., Ostareck-Lederer, A., Grskovic, M. & Hentze, M.W.
Cold Spring Harb Symp Quant Biol 2001;66:329-36. Europe PMC
Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system.
Bergamini, G., Preiss, T. & Hentze, M.W.
RNA 2000 Dec;6(12):1781-90.
In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting.
A new era for the RNA world. Conference: RNA 2000.
Hentze, M.W., Izaurralde, E. & Seraphin, B.
EMBO Rep. 2000 Nov;1(5):394-8. Europe PMC
The yeast nuclear cap binding complex can interact with translation factor eIF4G and mediate translation initiation.
Fortes, P., Inada, T., Preiss, T., Hentze, M.W., Mattaj, I.W. & Sachs, A.B.
Mol Cell 2000 Jul;6(1):191-6
The mRNA cap structure is bound by either the nuclear (CBC) or the cytoplasmic (eIF4F) cap binding complex. Following mRNA export, CBC must be exchanged for eIF4F in the cytoplasm. It is not known how this exchange occurs or how this RNP remodeling event is integrated with mRNA function. Here we report genetic and biochemical evidence that the yeast translation initiation factor eIF4G associates with CBC, and that eIF4E, the eIF4F component that binds both the cap and eIF4G, antagonizes this interaction. Furthermore, we find that CBC can stimulate translation in extracts containing an eIF4G protein deficient for eIF4E binding. These data suggest that eIF4E binding to the eIF4G-CBC complex on newly exported mRNA displaces CBC, and that the first round of translation on mRNA may occur via a different mechanism than subsequent rounds.
[Pair of siblings of Italian ethnicity with hyperferritinemia and cataract]
Volkmann, M., Schiff, J.H., Hor, M., Hentze, M.W., Fiehn, W. & Merkt, J.
Internist (Berl) 2000 Apr;41(4):381-4 Europe PMC
A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation.
McKie, A.T., Marciani, P., Rolfs, A., Brennan, K., Wehr, K., Barrow, D., Miret, S., Bomford, A., Peters, T.J., Farzaneh, F., Hediger, M.A., Hentze, M.W. & Simpson, R.J.
Mol Cell 2000 Feb;5(2):299-309
Iron absorption by the duodenal mucosa is initiated by uptake of ferrous Fe(II) iron across the brush border membrane and culminates in transfer of the metal across the basolateral membrane to the portal vein circulation by an unknown mechanism. We describe here the isolation and characterization of a novel cDNA (Ireg1) encoding a duodenal protein that is localized to the basolateral membrane of polarized epithelial cells. Ireg1 mRNA and protein expression are increased under conditions of increased iron absorption, and the 5' UTR of the Ireg1 mRNA contains a functional iron-responsive element (IRE). IREG1 stimulates iron efflux following expression in Xenopus oocytes. We conclude that IREG1 represents the long-sought duodenal iron export protein and is upregulated in the iron overload disease, hereditary hemochromatosis.
Translational control of developmental decisions.
Wickens, M., E.B. Goodwin, J. Kimble, S. Strickland & Hentze, M.W.
In "Translational Control of Gene Expression" Sonenberg, N., Hershey, J.W.B. & Mathews, M.B. (eds.) Cold Spring Harbor Press, pp. 295-370
Translational repression assay procedure: a method to study RNA-protein interactions in yeast.
Paraskeva, E. & Hentze, M.W.
Methods Enzymol. 2000;318:374-84. Europe PMC
Nitric oxide, oxygen radicals and iron metabolism.
Pantopoulos, K. & Hentze, M.W.
In "Nitric Oxide: Biology and Pathobiology", L. Ignarro (ed.). Academic Press, 293-313
Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries.
Castagnetti, S., Hentze, M.W., Ephrussi, A. & Gebauer, F.
The coupled regulation of oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of oskar mRNA, a mechanism whereby oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of oskar mRNA. We have established a cell- free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that this ovary extract, containing endogenous Bruno, is able to recapitulate oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, we show that a Drosophila embryo extract lacking Bruno efficiently translates oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in oskar mRNA translation. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.
HFE downregulates iron uptake from transferrin and induces iron- regulatory protein activity in stably transfected cells.
Riedel, H.D., Muckenthaler, M.U., Gehrke, S.G., Mohr, I., Brennan, K., Herrmann, T., Fitscher, B.A., Hentze, M.W. & Stremmel, W.
Blood 1999 Dec 1;94(11):3915-21
Hereditary hemochromatosis (HH) is a common autosomal-recessive disorder of iron metabolism. More than 80% of HH patients are homozygous for a point mutation in a major histocompatibility complex (MHC) class I type protein (HFE), which results in a lack of HFE expression on the cell surface. A previously identified interaction of HFE and the transferrin receptor suggests a possible regulatory role of HFE in cellular iron absorption. Using an HeLa cell line stably transfected with HFE under the control of a tetracycline-sensitive promoter, we investigated the effect of HFE expression on cellular iron uptake. We demonstrate that the overproduction of HFE results in decreased iron uptake from diferric transferrin. Moreover, HFE expression activates the key regulators of intracellular iron homeostasis, the iron-regulatory proteins (IRPs), implying that HFE can affect the intracellular "labile iron pool." The increase in IRP activity is accompanied by the downregulation of the iron-storage protein, ferritin, and an upregulation of transferrin receptor levels. These findings are discussed in the context of the pathophysiology of HH and a possible role of iron-responsive element (IRE)-containing mRNAs.
Translational control of dosage compensation in Drosophila by Sex- lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.
Gebauer, F., Corona, D.F., Preiss, T., Becker, P.B. & Hentze, M.W.
EMBO J 1999 Nov 1;18(21):6146-54
Translational repression of male-specific-lethal 2 (msl-2) mRNA by Sex- lethal (SXL) controls dosage compensation in Drosophila. In vivo regulation involves cooperativity between SXL-binding sites in the 5' and 3' untranslated regions (UTRs). To investigate the mechanism of msl- 2 translational control, we have developed a novel cell-free translation system from Drosophila embryos that recapitulates the critical features of mRNA translation in eukaryotes: cap and poly(A) tail dependence. Importantly, tight regulation of msl-2 translation in this system requires cooperation between the SXL-binding sites in both the 5' and 3' UTRs, as seen in vivo. However, in contrast to numerous other developmentally regulated mRNAs, the regulation of msl-2 mRNA occurs by a poly(A) tail-independent mechanism. The approach described here allows mechanistic analysis of translational control in early Drosophila development and has revealed insights into the regulation of dosage compensation by SXL.
Human cytoplasmic aconitase (Iron regulatory protein 1) is converted into its [3Fe-4S] form by hydrogen peroxide in vitro but is not activated for iron-responsive element binding.
Brazzolotto, X., Gaillard, J., Pantopoulos, K., Hentze, M.W. & Moulis, J.M.
J Biol Chem 1999 Jul 30;274(31):21625-30
Iron regulatory protein 1 (IRP1) regulates the synthesis of proteins involved in iron homeostasis by binding to iron-responsive elements (IREs) of messenger RNA. IRP1 is a cytoplasmic aconitase when it contains a [4Fe-4S] cluster and an RNA-binding protein after complete removal of the metal center by an unknown mechanism. Human IRP1, obtained as the pure recombinant [4Fe-4S] form, is an enzyme as efficient toward cis-aconitate as the homologous mitochondrial aconitase. The aconitase activity of IRP1 is rapidly lost by reaction with hydrogen peroxide as the [4Fe-4S] cluster is quantitatively converted into the [3Fe-4S] form with release of a single ferrous ion per molecule. The IRE binding capacity of IRP1 is not elicited with H(2)O(2). Ferrous sulfate (but not other more tightly coordinated ferrous ions, such as the complex with ethylenediamine tetraacetic acid) counteracts the inhibitory action of hydrogen peroxide on cytoplasmic aconitase, probably by replenishing iron at the active site. These results cast doubt on the ability of reactive oxygen species to directly increase IRP1 binding to IRE and support a signaling role for hydrogen peroxide in the posttranscriptional control of proteins involved in iron homeostasis in vivo.
Ligand-induced structural alterations in human iron regulatory protein- 1 revealed by protein footprinting.
Gegout, V., Schlegl, J., Schlager, B., Hentze, M.W., Reinbolt, J., Ehresmann, B., Ehresmann, C. & Romby, P.
J Biol Chem 1999 May 21;274(21):15052-8
Human iron regulatory protein-1 (IRP-1) is a bifunctional protein that regulates iron metabolism by binding to mRNAs encoding proteins involved in iron uptake, storage, and utilization. Intracellular iron accumulation regulates IRP-1 function by promoting the assembly of an iron-sulfur cluster, conferring aconitase activity to IRP-1, and hindering RNA binding. Using protein footprinting, we have studied the structure of the two functional forms of IRP-1 and have mapped the surface of the iron-responsive element (IRE) binding site. Binding of the ferritin IRE or of the minimal regulatory region of transferrin receptor mRNA induced strong protections against proteolysis in the region spanning amino acids 80 to 187, which are located in the putative cleft thought to be involved in RNA binding. In addition, IRE- induced protections were also found in the C-terminal domain at Arg-721 and Arg-728. These data implicate a bipartite IRE binding site located in the putative cleft of IRP-1. The aconitase form of IRP-1 adopts a more compact structure because strong reductions of cleavage were detected in two defined areas encompassing residues 149 to 187 and 721 to 735. Thus both ligands of apo-IRP-1, the IRE and the 4Fe-4S cluster, induce distinct but overlapping alterations in protease accessibility. These data provide evidences for structural changes in IRP-1 upon cluster formation that affect the accessibility of residues constituting the RNA binding site.
Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress.
Gehring, N.H., Hentze, M.W. & Pantopoulos, K.
J Biol Chem 1999 Mar 5;274(10):6219-25
Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H2O2. Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1. Treatment of murine B6 fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, causes a modest activation of IRP-1 to bind to IREs within 15-30 min. However, IRE binding drops to basal levels within 60 min. Surprisingly, a remarkable loss of both IRE binding and aconitase activities of IRP-1 follows treatment with MSB for 1-2 h. These effects do not result from alterations in IRP-1 half-life, can be antagonized by the antioxidant N- acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron- starved MSB-treated cells to increase transferrin receptor mRNA levels is inhibited, and MSB increases the translation of a human growth hormone indicator mRNA bearing an IRE in its 5'-untranslated region. Nonetheless, MSB inhibits ferritin synthesis. Thus, menadione-induced oxidative stress leads to post-translational inactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the "classical" Fe-S cluster switch and exerts multiple effects on cellular iron metabolism.
A perfect message: RNA surveillance and nonsense-mediated decay.
Hentze, M.W. & Kulozik, A.E.
Cell 1999 Feb 5;96(3):307-10. Europe PMC
Ribosomal pausing and scanning arrest as mechanisms of translational regulation from cap-distal iron-responsive elements.
Paraskeva, E., Gray, N.K., Schlager, B., Wehr, K. & Hentze, M.W.
Mol Cell Biol 1999 Jan 19(1) 807-816
Iron regulatory protein 1 (IRP-1) binding to an iron-responsive element (IRE) located close to the cap structure of mRNAs represses translation by precluding the recruitment of the small ribosomal subunit to these mRNAs. This mechanism is position dependent; reporter mRNAs bearing IREs located further downstream exhibit diminished translational control in transfected mammalian cells. To investigate the underlying mechanism, we have recapitulated this position effect in a rabbit reticulocyte cell-free translation system. We show that the recruitment of the 43S preinitiation complex to the mRNA is unaffected when IRP-1 is bound to a cap-distal IRE. Following 43S complex recruitment, the translation initiation apparatus appears to stall, before linearly progressing to the initiation codon. The slow passive dissociation rate of IRP-1 from the cap-distal IRE suggests that the mammalian translation apparatus plays an active role in overcoming the cap-distal IRE-IRP-1 complex. In contrast, cap-distal IRE-IRP-1 complexes efficiently repress translation in wheat germ and yeast translation extracts. Since inhibition occurs subsequent to 43S complex recruitment, an efficient arrest of productive scanning may represent a second mechanism by which RNA-protein interactions within the 5' untranslated region of an mRNA can regulate translation. In contrast to initiating ribosomes, elongating ribosomes from mammal, plant, and yeast cells are unaffected by IRE-IRP-1 complexes positioned within the open reading frame. These data shed light on a characteristic aspect of the IRE-IRP regulatory system and uncover properties of the initiation and elongation translation apparatus of eukaryotic cells.
Regulation of iron metabolism in higher eukaryotes: iron-sulfur centers as genetic switches.
Pantopoulos, K. & Hentze, M.W.
In "Inorganic biochemistry and regulatory mechanisms of iron metabolism", Ferreira, G. C., Moura, J. J. G. & Franco, R. (eds.), Wiley-VCH, Weinheim, pp. 131-152
Poly(A)-tail-promoted translation in yeast: implications for translational control.
Preiss, T., Muckenthaler, M. & Hentze, M.W.
RNA 1998 Nov 4(11) 1321-1331
The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo. Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA. This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis. We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs. Both manipulations effectively block cap- dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks. We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements. With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs. We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions. Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.
Cytoplasmic regulatory functions of the KH-domain proteins hnRNPs K and E1/E2.
Ostareck-Lederer, A., Ostareck, D.H. & Hentze, M.W.
This is a review article.
Trends Biochem Sci 1998 Nov 23(11) 409-411 Europe PMC
Activation of iron regulatory protein-1 by oxidative stress in vitro.
Pantopoulos, K. & Hentze, M.W.
Proc Natl Acad Sci U S A 1998 Sep 1 95(18) 10559-10563
Iron regulatory protein-1 (IRP-1), a central cytoplasmic regulator of cellular iron metabolism, is rapidly activated by oxidative stress to bind to mRNA iron-responsive elements. We have reconstituted the response of IRP-1 to extracellular H2O2 in a system derived from murine B6 fibroblasts permeabilized with streptolysin-O. This procedure allows separation of the cytosol from the remainder of the cells (cell pellet). IRP-1 in the cytosolic fraction fails to be directly activated by addition of H2O2. IRP-1 activation requires the presence of a nonsoluble, possibly membrane-associated component in the cell pellet. The streptolysin-O-based in vitro system faithfully recapitulates characteristic hallmarks of IRP-1 activation by H2O2 in intact cells. We show that the H2O2-mediated activation of IRP-1 is temperature dependent and sensitive to treatment with calf intestinal alkaline phosphatase (CIAP). Although IRP-1 activation is unaffected by addition of excess ATP or GTP to this in vitro system, it is negatively affected by the nonhydrolyzable nucleotide analogs adenylyl-imidodiphosphate and guanylyl-imidophosphate and completely blocked by ATP-gammaS and GTP- gammaS. The in vitro reconstitution of this oxidative stress-induced pathway has opened a different avenue for the biochemical dissection of the regulation of mammalian iron metabolism by oxidative stress. Our data show that H2O2 must be sensed to stimulate a pathway to activate IRP-1.
IRP-1 binding to ferritin mRNA prevents the recruitment of the small ribosomal subunit by the cap-binding complex eIF4F.
Muckenthaler, M., Gray, N.K. & Hentze, M.W.
Mol Cell 1998 Sep 2(3) 383-388
Binding of iron regulatory proteins (IRPs) to IREs located in proximity to the cap structure of ferritin H- and L-chain mRNAs blocks ferritin synthesis by preventing the recruitment of the small ribosomal subunit to the mRNA. We have devised a novel procedure to examine the assembly of translation initiation factors (eIFs) on regulated mRNAs. Unexpectedly, we find that the cap binding complex eIF4F (comprising eIF4E, eIF4G, and eIF4A) assembles even when IRP-1 is bound to the cap- proximal IRE. This assembly is futile, because bridging interactions between eIF4F and the small ribosomal subunit cannot be established in the presence of IRP-1. Our findings provide insight into translational control by an mRNA binding protein at the level of translation initiation factors and uncover a key regulatory step in iron homeostasis.
Molecular cloning and characterisation of a novel duodenal-specific gene implicated in iron absorption.
McKie, A.T., Wehr, K., Simpson, R.J., Peters, T.J., Hentze, M.W. & Farzaneh, F.
Biochem Soc Trans 1998 Aug;26(3):S264 Europe PMC
Translational activation of uncapped mRNAs by the central part of human eIF4G is 5' end-dependent.
De Gregorio, E., Preiss, T. & Hentze, M.W.
RNA 1998 Jul 4(7) 828-836
Translation initiation factor (eIF) 4G represents a critical link between mRNAs and 40S ribosomal subunits during translation initiation. It interacts directly with the cap-binding protein eIF4E through its N- terminal part, and binds eIF3 and eIF4A through the central and C- terminal region. We expressed and purified recombinant variants of human eIF4G lacking the N-terminal domain as GST-fusion proteins, and studied their function in cell-free translation reactions. Both eIF4G lacking its N-terminal part (aa 486-1404) and the central part alone (aa 486-935) exert a dominant negative effect on the translation of capped mRNAs. Furthermore, these polypeptides potently stimulate the translation of uncapped mRNAs. Although this stimulation is cap- independent, it is shown to be dependent on the accessibility of the mRNA 5' end. These results reveal two unexpected features of eIF4G- mediated translation. First, the C-terminal eIF4A binding site is dispensable for activation of uncapped mRNA translation. Second, translation of uncapped mRNA still requires 5' end-dependent ribosome binding. These new findings are incorporated into existing models of mammalian translation initiation.
Binary specification of nonsense codons by splicing and cytoplasmic translation.
Thermann, R., Neu-Yilik, G., Deters, A., Frede, U., Wehr, K., Hagemeier, C., Hentze, M.W. & Kulozik, A.E.
EMBO J 1998 Jun 15 17(12) 3484-3494
Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.
Dual function of the messenger RNA cap structure in poly(A)-tail- promoted translation in yeast.
Preiss, T. & Hentze, M.W.
Nature 1998 Apr 2 392(6675) 516-520
The messenger RNA 3' poly(A) tail critically affects the initiation and control of translation in eukaryotes. By analogy to elements involved in transcription initiation, the poly(A) tail has been described as a 'translational enhancer' that enhances the 'translational promoter' activity of the mRNA 5'-cap structure. Elongation or shortening of the poly(A) tail regulates translation during development. Here we show, using cell-free and in vivo translation analyses in Saccharomyces cerevisiae, that the poly(A) tail can act as an independent 'translational promoter', delivering ribosomes to uncapped mRNAs even if their 5' end is blocked. When mRNAs compete for ribosome binding, neither the cap structure nor the poly(A) tail alone is enough to drive efficient translation, but together they synergize and direct ribosome entry to the 5' end. The cap structure both promotes ribosome recruitment, together with the poly(A) tail, and tethers recruited ribosomes to the 5' end. Correct choice of translation initiation codons and the function of translational regulators acting on the 5' untranslated region are thus ensured by the functional interaction of the poly(A) tail with the cap structure.
A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo.
Paraskeva, E., Atzberger, A. & Hentze, M.W.
Proc Natl Acad Sci U S A 1998 Feb 3 95(3) 951-956
RNA-protein interactions are central to many aspects of cellular metabolism, cell differentiation, and development as well as the replication of infectious pathogens. We have devised a versatile, broadly applicable in vivo system for the analysis of RNA-protein interactions in yeast. TRAP (translational repression assay procedure) is based on the translational repression of a reporter mRNA encoding green fluorescent protein by an RNA-binding protein for which a cognate binding site has been introduced into the 5' untranslated region. Because protein binding to the 5' untranslated region can sterically inhibit ribosome association, expression of the cognate binding protein causes significant reduction in the levels of green fluorescent protein fluorescence. By using RNA-protein interactions with affinities in the micromolar to nanomolar range, we demonstrate the specificity of TRAP as well as its ability to recover the cDNA encoding a specific RNA- binding protein, which has been diluted 500,000-fold with unrelated cDNAs, by using fluorescence-activated cell sorting. We suggest that TRAP offers a strategy to clone RNA-binding proteins for which little else than the binding site is known, to delineate RNA sequence requirements for protein binding as well as the protein domains required for RNA binding, and to study effectors of RNA-protein interactions in vivo.
The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA.
Gebauer, F., Merendino, L., Hentze, M.W. & Valcarcel, J.
RNA 1998 Feb 4(2) 142-150
Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5' UTR of msl-2 mRNA resulted in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.
Iron-regulatory protein-1 (IRP-1) is highly conserved in two invertebrate species--characterization of IRP-1 homologues in Drosophila melanogaster and Caenorhabditis elegans.
Muckenthaler, M., Gunkel, N., Frishman, D., Cyrklaff, A., Tomancak, P. & Hentze, M.W.
Eur J Biochem. 1998 Jun 1;254(2):230-7.
Iron-regulatory protein-1 (IRP-1) plays a dual role as a regulatory RNA-binding protein and as a cytoplasmic aconitase. When bound to iron-responsive elements (IRE), IRP-1 post-transcriptionally regulates the expression of mRNAs involved in iron metabolism. IRP have been cloned from several vertebrate species. Using a degenerate-primer PCR strategy and the screening of data bases, we now identify the homologues of IRP-1 in two invertebrate species, Drosophila melanogaster and Caenorhabditis elegans. Comparative sequence analysis shows that these invertebrate IRP are closely related to vertebrate IRP, and that the amino acid residues that have been implicated in aconitase function are particularly highly conserved, suggesting that invertebrate IRP may function as cytoplasmic aconitases. Antibodies raised against recombinant human IRP-1 immunoprecipitate the Drosophila homologue expressed from the cloned cDNA. In contrast to vertebrates, two IRP-1 homologues (Drosophila IRP-1A and Drosophila IRP-1B), displaying 86% identity to each other, are expressed in D. melanogaster. Both of these homologues are distinct from vertebrate IRP-2. In contrast to the mammalian system where the two IRP (IRP-1 and IRP-2) are differentially expressed, Drosophila IRP-1A and Drosophila IRP-1B are not preferentially expressed in specific organs. The localization of Drosophila IRP-1A to position 94C1-8 and of Drosophila IRP-1B to position 86B3-6 on the right arm of chromosome 3 and the availability of an IRP-1 cDNA from C. elegans will facilitate a genetic analysis of the IRE/IRP system, thus opening a new avenue to explore this regulatory network.
Systematic genomic screening and analysis of mRNA in untranslated regions and mRNA precursors: combining experimental and computational approaches.
Dandekar, T., Beyer, K., Bork, P., Kenealy, M.R., Pantopoulos, K., Hentze, M., Sonntag-Buck, V., Flouriot, G., Gannon, F. & Schreiber, S.
MOTIVATION: The untranslated regions (UTRs) of mRNA upstream (5'UTR) and downstream (3'UTR) of the open reading frame, as well as the mRNA precursor, carry important regulatory sequences. To reveal unidentified regulatory signals, we combine information from experiments with computational approaches. Depending on available knowledge, three different strategies are employed. RESULTS: Searching with a consensus template, new RNAs with regulatory RNA elements can be identified in genomic screens. By this approach, we identify new candidate regulatory motifs resembling iron-responsive elements in the 5'UTRs of HemA, FepB and FrdB mRNA from Escherichia coli. If an RNA element is not yet defined, it may be analyzed by combining results from SELEX (selective enrichment of ligands by exponential amplification) and a search of databases from RNA or genomic sequences. A cleavage stimulating factor (CstF) binding element 3 of the polyadenylation site in the mRNA precursor serves as a test example. Alternatively, the regulatory RNA element may be found by studying different RNA foldings and their correlation with simple experimental tests. We delineate a novel instability element in the 3'UTR of the estrogen receptor mRNA in this way. AVAILABILITY: Strategy, methods and programs are available on request from T.Dandekar. CONTACT: email@example.com
Novel functions for 'nuclear factors' in the cytoplasm: the Sex-lethal paradigm.
Gebauer, F., Merendino, L., Hentze, M.W. & Valcarcel, J.
Semin Cell Dev Biol 1997 Dec 8(6) 561-566
In recent years, novel functions for a number of nuclear factors have been uncovered in the cytoplasm, mainly at the level of translation. These factors behave as multifunctional regulators of gene expression and many play key roles in cell differentiation and development. One of the best characterized examples is that of Sex-lethal (SXL), an RNA- binding protein that is expressed in female Drosophila flies and controls sex determination and dosage compensation. Recent findings indicate that SXL, a paradigmatic regulator of splicing, also controls translation of target mRNAs. This review attempts to summarize this evidence and provide an overview of 'nuclear factors' with roles in translation.Copyright 1998 Academic Press Limited
Probing the structure of the regulatory region of human transferrin receptor messenger RNA and its interaction with iron regulatory protein- 1.
Schlegl, J., Gegout, V., Schlager, B., Hentze, M.W., Westhof, E., Ehresmann, C., Ehresmann, B. & Romby, P.
RNA 1997 Oct;3(10):1159-72
A portion of the 3'UTR of the human transferrin receptor mRNA mediates iron-dependent regulation of mRNA stability. The minimal RNA regulatory region contains three conserved hairpins, so-called iron responsive elements (IREs), that are recognized specifically by iron regulatory proteins (IRPs). The structure of this regulatory region and its complex with IRP-1 was probed using a combination of enzymes and chemicals. The data support the existence of an intrinsic IRE loop structure that is constrained by an internal C-G base pair. This particular structure is one of the determinants required for optimal IRP binding. IRP-1 covers one helical turn of the IRE and protects conserved residues in each of the three IREs: the bulged cytosine and nucleotides in the hairpin loops. Two essential IRP-phosphate contacts were identified by ethylation interference. Three-dimensional modeling of one IRE reveals that IRP-1 contacts several bases and the ribose- phosphate backbone located on one face in the deep groove, but contacts also exist with the shallow groove. A conformational change of the IRE loop mediated by IRP-1 binding was visualized by Pb2+-catalyzed hydrolysis. This effect is dependent on the loop structure and on the nature of the closing base pair. Within the regulatory region of transferrin receptor mRNA, IRP-1 induces reactivity changes in a U-rich hairpin loop that requires the presence of the stem-loop structure located just downstream the endonucleolytic cleavage site identified by Binder et al. (Binder R et al. 1994, EMBO J 13:1969-1980). These results provide indications of the mechanism by which IRP-1 stabilizes the transferrin receptor mRNA under iron depletion conditions.
Regulated poly(A) tail shortening in somatic cells mediated by cap- proximal translational repressor proteins and ribosome association.
Muckenthaler, M., Gunkel, N., Stripecke, R. & Hentze, M.W.
RNA 1997 Sep;3(9):983-95
The poly(A) tail plays an important role in translation initiation. We report the identification of a mechanism that operates in mammalian somatic cells, and couples mRNA poly(A) tail length with its translation state. The regulation of human ferritin L-chain mRNA by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) is subject to this mechanism: translational repression imposed by IRP binding to the IRE of ferritin L-chain mRNA induces poly(A) tail shortening. For the accumulation of mRNAs with short poly(A) tails, IRP binding to an IRE per se is not sufficient, but must cause translational repression. Interestingly, puromycin and verrucarin (general translation inhibitors that dissociate mRNAs from ribosomes) mimick the negative effect of the specific translational repressor proteins on poly(A) tail length, whereas cycloheximide and anisomycin (general translation inhibitors that maintain the association between mRNAs and ribosomes) preserve long poly(A) tails. Thus, the ribosome association of the mRNA appears to represent the critical determinant. These findings identify a novel mechanism of regulated polyadenylation as a consequence of translational control. They reveal differences in poly(A) tail metabolism between polysomal and mRNP-associated mRNAs. A possible role of this mechanism in the maintenance of translational repression is discussed.
Starting at the beginning, middle, and end: translation initiation in eukaryotes.
Sachs, A.B., Sarnow, P. & Hentze, M.W.
This is a review article.
Cell 1997 Jun 13 89(6) 831-838 Europe PMC
mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end.
Ostareck, D.H., Ostareck-Lederer, A., Wilm, M., Thiele, B.J., Mann, M. & Hentze, M.W.
Cell 1997 May 16;89(4):597-606
Although LOX mRNA accumulates early during differentiation, a differentiation control element in its 3' untranslated region confers translational silencing until late stage erythropoiesis. We have purified two proteins from rabbit reticulocytes that specifically mediate LOX silencing and identified them as hnRNPs K and E1. Transfection of hnRNP K and hnRNP E1 into HeLa cells specifically silenced the translation of reporter mRNAs bearing a differentiation control element in their 3' untranslated region. Silenced LOX mRNA in rabbit reticulocytes specifically coimmunoprecipitated with hnRNP K. In a reconstituted cell-free translation system, addition of recombinant hnRNP K and hnRNP E1 recapitulates this regulation via a specific inhibition of 80S ribosome assembly on LOX mRNA. Both proteins can control cap-dependent and internal ribosome entry site-mediated translation by binding to differentiation control elements. Our data suggest a specific cytoplasmic function for hnRNPs as translational regulatory proteins.
Differences in the regulation of iron regulatory protein-1 (IRP-1) by extra- and intracellular oxidative stress.
Pantopoulos, K., Mueller, S., Atzberger, A., Ansorge, W., Stremmel, W. & Hentze, M.W.
J Biol Chem 1997 Apr 11;272(15):9802-8
We have studied the responses of iron regulatory protein-1 (IRP-1) to extra- and intracellular sources of reactive oxygen intermediates (ROIs). IRP-1 is a cytoplasmic RNA-binding protein that regulates iron metabolism following its activation by iron deficiency, nitric oxide, and administration of H2O2 or antimycin A, an inhibitor of the respiratory chain (Hentze, M. W., and Kuhn, L. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8175-8182). We show that 10 &mgr;M H2O2 suffice for complete IRP-1 activation within 60 min when H2O2 is generated extracellularly at steady-state. By contrast, rapid cellular H2O2 degradation necessitates a 5-10-fold higher bolus dose. To study IRP-1 responses to intracellular oxidative stress, mitochondrial respiration was inhibited with antimycin A (to generate oxidative stress by leakage of ROIs from complex III), or catalase was blocked with 3-amino-1,2,4- triazole (to diminish H2O2 degradation); in parallel, 2',7'- dichlorodihydrofluorescein diacetate was used as a redox-sensitive probe to monitor intracellular H2O2 levels by fluorescence-activated cell sorting. Catalase inhibition elevates intracellular H2O2, but surprisingly does not cause concomitant IRP-1 activation. Following antimycin A treatment, IRP-1 is activated, but the activation kinetics lag behind the rapid increase in detectable intracellular H2O2. IRP-1 is thus activated both by extra- and intracellular generation of ROIs. While extracellular H2O2 rapidly activates IRP-1 even without detectable increases in intracellular H2O2, intracellular H2O2 elevation is not sufficient for IRP-1 activation. IRP-1 thus represents a novel example of an H2O2-regulated protein that responds differentially to alterations of extra- and intracellular H2O2 levels. Our data also suggest that a direct attack on the 4Fe-4S cluster of IRP- 1 by H2O2 (or an H2O2-derived reactive species) represents an unlikely explanation for IRP-1 activation by oxidative stress.
eIF4G: a multipurpose ribosome adapter?
This is a review article.
Science 1997 Jan 24 275(5299) 500-501 Europe PMC
Pathways for the regulation of macrophage iron metabolism by the anti- inflammatory cytokines IL-4 and IL-13.
Weiss, G., Bogdan, C. & Hentze, M.W.
J Immunol 1997 Jan 1;158(1):420-5
Macrophage effector functions are influenced by their iron status and by shifts in the balance between type 1 Th1 and Th2 cells. To elucidate the influence of the Th2 cytokines IL-4 and IL-13 on macrophage iron metabolism, we investigated activated primary mouse macrophages and the murine macrophage cell line J774. Stimulation of J774 cells and primary macrophages with IFN-gamma/LPS activates the RNA binding affinities of iron regulatory protein-1 (IRP-1) and IRP-2 for iron-responsive elements, leading to translational repression of the iron storage protein ferritin. Activation of IRP-1 and IRP-2 is caused by increased formation of nitric oxide (NO) via stimulation of the inducible NO synthase by IFN-gamma/LPS. Treatment of macrophages with IL-4 and/or IL- 13 before stimulation with IFN-gamma/LPS suppresses NO formation and IRP activation, with concomitantly enhanced ferritin synthesis despite a small reduction in ferritin heavy chain mRNA levels. The mRNA levels for the membrane receptor for iron uptake, transferrin receptor (TfR), decrease following stimulation with IFN-gamma/LPS, although IRP- mediated stabilization of the TfR mRNA would have been expected. This as yet unidentified proximal inhibitory signal by IFN-gamma/LPS is antagonized by IL-4 and/or IL-13, which leads to increased TfR mRNA expression in an IRP-independent manner. Thus, IL-4 and IL-13 regulate the iron metabolism of activated macrophages by at least two different pathways: first, by opposing NO-mediated IRP activation, thereby increasing ferritin translation; and second, by an IRP-independent augmentation of TfR mRNA expression. We suggest that IL-4 and IL-13 may enhance iron uptake and storage in activated macrophages and thereby contribute to down-regulation of macrophage effector functions.
Iron regulatory protein-1 (IRP-1) is differentially regulated by extracellular and intracellular hydrogen-peroxide.
Muller, S, Pantopoulas, K., Atzberger, A., Ansorge, W., Hentze, M.W. & Stremmel, W.
Gastroenterology 1997 112(4) 419
Mechanisms for posttranscriptional regulation by iron-responsive elements and iron regulatory proteins.
Muckenthaler, M. & Hentze, M.W.
This is a review article.
Prog Mol Subcell Biol 1997;18:93-115 Europe PMC
Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress.
Hentze, M.W. & Kuhn, L.C.
This is a review article.
Proc Natl Acad Sci U S A 1996 Aug 6;93(16):8175-82
As an essential nutrient and a potential toxin, iron poses an exquisite regulatory problem in biology and medicine. At the cellular level, the basic molecular framework for the regulation of iron uptake, storage, and utilization has been defined. Two cytoplasmic RNA-binding proteins, iron-regulatory protein-1 (IRP-1) and IRP-2, respond to changes in cellular iron availability and coordinate the expression of mRNAs that harbor IRP-binding sites, iron-responsive elements (IREs). Nitric oxide (NO) and oxidative stress in the form of H2O2 also signal to IRPs and thereby influence cellular iron metabolism. The recent discovery of two IRE-regulated mRNAs encoding enzymes of the mitochondrial citric acid cycle may represent the beginnings of elucidating regulatory coupling between iron and energy metabolism. In addition to providing insights into the regulation of iron metabolism and its connections with other cellular pathways, the IRE/IRP system has emerged as a prime example for the understanding of translational regulation and mRNA stability control. Finally, IRP-1 has highlighted an unexpected role for iron sulfur clusters as post-translational regulatory switches.
Iron-sulfur clusters and oxidant stress responses
Trends Biochem Sci 1996 Aug;21(8):282-3 Europe PMC
Conservation of aconitase residues revealed by multiple sequence analysis. Implications for structure/function relationships.
Frishman, D. & Hentze, M.W.
Eur J Biochem 1996 Jul 1;239(1):197-200
Aconitases have recently regained much attention, because one member of this family, iron regulatory protein-1 (IRP-1), has been found to play a dual role as a cytoplasmic aconitase and a regulatory RNA-binding protein. This finding has highlighted a novel role for Fe-S clusters as post-translational regulatory switches. We have aligned 28 members of the Fe-S isomerase family, identified highly conserved amino acid residues, and integrated this information with data on the crystallographic structure of mammalian mitochondrial aconitase. We propose structural and/or functional roles for the previously unrecognized conserved residues. Our findings illustrate the value of detailed protein sequence analysis when high-resolution crystallographic data are already available.
Nitric oxide and oxidative stress (H2O2) control mammalian iron metabolism by different pathways.
Pantopoulos, K., Weiss, G. & Hentze, M.W.
Mol Cell Biol 1996 Jul;16(7):3781-8
Several cellular mRNAs are regulated posttranscriptionally by iron- responsive elements (IREs) and the cytosolic IRE-binding proteins IRP-1 and IRP-2. Three different signals are known to elicit IRP-1 activity and thus regulate IRE-containing mRNAs: iron deficiency, nitric oxide (NO), and the reactive oxygen intermediate hydrogen peroxide (H2O2). In this report, we characterize the pathways for IRP-1 regulation by NO and H2O2 and examine their effects on IRP-2. We show that the responses of IRP-1 and IRP-2 to NO remarkably resemble those elicited by iron deficiency: IRP-1 induction by NO and by iron deficiency is slow and posttranslational, while IRP-2 induction by these inductive signals is slow and requires de novo protein synthesis. In contrast, H2O2 induces a rapid posttranslational activation which is limited to IRP-1. Removal of the inductive signal H2O2 after < or = 15 min of treatment (induction phase) permits a complete IRP-1 activation within 60 min (execution phase) which is sustained for several hours. This contrasts with the IRP-1 activation pathway by NO and iron depletion, in which NO- releasing drugs or iron chelators need to be present during the entire activation phase. Finally, we demonstrate that biologically synthesized NO regulates the expression of IRE-containing mRNAs in target cells by passive diffusion and that oxidative stress endogenously generated by pharmacological modulation of the mitochondrial respiratory chain activates IRP-1, underscoring the physiological significance of NO and reactive oxygen intermediates as regulators of cellular iron metabolism. We discuss models to explain the activation pathways of IRP- 1 and IRP-2. In particular, we suggest the possibility that NO affects iron availability rather than the iron-sulfur cluster of IRP-1.
Iron-sulphur clusters as genetic regulatory switches: the bifunctional iron regulatory protein-1.
Paraskeva, E. & Hentze, M.W.
This is a review article.
FEBS Lett 1996 Jun 24;389(1):40-3
In the eighties, iron regulatory protein-1 (IRP-1) was identified as a cytoplasmic mRNA-binding protein that regulates vertebrate cell iron metabolism. More recently, IRP-1 was found to represent the functional cytoplasmic homologue of mitochondrial aconitase, a citric acid cycle enzyme. Its two functions are mutually exclusive and depend on the status of an Fe-S cluster: the (cluster-less) apoIRP-1 binds to RNA, while the incorporation of a cubane 4Fe-4S cluster is required for enzymatic activity. Cellular signals including iron levels, nitric oxide and oxidative stress can regulate between the two activities posttranslationally and reversibly via the Fe-S cluster. Recent reports suggest that other regulatory proteins may be controlled by similar mechanisms.
Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements.
Gray, N.K., Pantopoulous, K., Dandekar, T., Ackrell, B.A. & Hentze, M.W.
Proc Natl Acad Sci U S A 1996 May 14;93(10):4925-30
The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell- free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.
Regulated ribosomal frameshifting by an RNA-protein interaction.
Kollmus, H., Hentze, M.W. & Hauser, H.
RNA 1996 Apr;2(4):316-23
Ribosomal frameshifting is a translational mechanism used as an essential step in the replication cycle of retroviruses. Programmed frameshifting in retroviral translation involves two sequence elements: A heptanucleotide slippery sequence which induces a low basal level of frameshifting and a downstream RNA structure as an enhancer of the process. The precise mechanism of function of these downstream elements is still unclear, but their effect does not solely depend on their stability. Likewise, the possibility that frameshifting could be controlled by specific proteins that bind to these elements and enable or modulate their effects has yet not been substantiated. The RNA hairpin of the HIV-1 gag-pol frameshift cassette was replaced by the iron-responsive element (IRE) from ferritin mRNA, a stem-loop structure that binds iron regulatory proteins (IRPs) in dependence of the iron status of the cell. When a lacZ/luciferase reporter construct was expressed in transfected BHK-21 cells, the IRE or a point-mutated version that is unable to bind IRPs were found to functionally substitute for the HIV-1 hairpin. When cells were treated with the iron chelator desferrioxamine to stimulate IRP binding to the wild-type IRE, frameshift activity was specifically and strongly augmented by protein binding Our data establish that frameshifting can be regulated in a reversible fashion by mRNA-binding proteins.
Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution.
Picard, V., Renaudie, F., Porcher, C., Hentze, M.W., Grandchamp, B. & Beaumont, C.
Blood 1996 Mar 1;87(5):2057-64
To test the hypothesis that variations in H- and L-subunit composition in the ferritin shell affect intracellular iron metabolism, we established stable transfectants of mouse erythroleukemia cells overexpressing the H-ferritin subunit. Analyses were performed on individual clones of transfected cells induced to differentiate with hexamethylenbisacetamide (HMBA). The results showed that there was a reduction in the amount of hemoglobin produced, in inverse relationship with the level of H-subunit overexpression. Incorporation of [2- 14C]glycine into heme was reduced by 20% t0 30% in the clones overexpressing H-ferritin subunit compared with control clone. However, the reduction in hemoglobin production was not reversed by addition of heme precursors (delta-aminolevulinic acid or iron) or by hemin itself. A reduced accumulation of beta-globin mRNA was also observed, which could account for the impaired hemoglobin synthesis. Furthermore, synthesis of the endogenous L-ferritin subunit was greatly repressed. Gel retardation assays performed on cytoplasmic extracts of transfected cells using an iron-responsive element (IRE) as a probe revealed that in overexpressing cells, the iron-regulatory protein (IRP) had a conformation with a high RNA-binding affinity, thus leading to translational repression of the endogenous L-ferritin synthesis. These data suggest that an increased formation of H-rich isoferritins leads to a rapid chelation of the regulatory iron pool. While the mechanism underlying the reduction in beta-globin mRNA remains to be elucidated, this study provides direct evidence for the role of IRP-mediated regulation of ferritin expression in erythroid cell metabolism.
Rapid responses to oxidative stress mediated by iron regulatory protein.
Pantopoulos, K. & Hentze, M.W.
EMBO J 1995 Jun 15;14(12):2917-24
Reactive oxygen intermediates (ROIs), including superoxide anion (O2.-) and hydrogen peroxide (H2O2), are by-products of aerobic metabolism with potential toxicity towards cellular macromolecules, including lipids, proteins and DNA. Excess ROIs, a condition referred to as oxidative stress, is considered to be a major contributor to ageing, degenerative diseases and reperfusion injury. The reactivity of H2O2 with iron (Fenton reaction) intimately connects oxidative stress and cellular iron metabolism. We have found a novel oxidative stress response pathway in mammalian cells which links oxidative stress to the regulation of iron metabolism. Exposure of cells to H2O2 leads to reduced synthesis of the intracellular iron storage protein ferritin and stimulates transferrin receptor (TfR) mRNA expression. Both responses are post-transcriptional and result from induction of iron regulatory protein (IRP) binding to iron-responsive elements (IREs) in ferritin and TfR mRNAs. IRP induction by H2O2 appears to involve the disassembly of its cubane 4Fe-4S cluster and occurs even in the presence of the protein synthesis inhibitor cycloheximide. The induction kinetics by H2O2 far exceed those by iron starvation. The response requires cellular integrity and cannot be elicited in cell extracts. Whereas the activation of IRP by iron depletion is insensitive to okadaic acid, the rapid induction by H2O2 is blocked by this inhibitor of type I/IIa protein phosphatases. Thus okadaic acid separates the activation pathways by iron depletion and oxidative stress, suggesting the involvement of stress-induced kinase/phosphatase pathways in the latter.