Niels Gehring, PhD 
Director's Research
EMBL
Meyerhofstrasse 1
D-69117 Heidelberg
Germany
Phone: +49-6221-564-579
Fax: +49-6221-387-8518
email: gehring@embl.de
Analysis of exon-junction complexes and the molecular mechanism of nonsense-mediated mRNA decay
Mammalian gene expression starts off in the nucleus with the transcription of the primary transcript, the precursor mRNA (pre-mRNA). The pre-mRNA then has to undergo several steps of processing before the mature, completely and correctly processed mRNA is exported to the cytoplasm, where it serves as template for protein biosynthesis. During processing of pre-mRNAs in the nucleus, the spliceosome loads the EJC (exon-junction complex) onto processed mRNAs. The exon junction complex plays a decisive role in nonsense-mediated mRNA decay (NMD), a cellular quality control mechanism eliminating nonsense-mutated mRNAs.
In addition, EJCs have been shown to enhance polysome association and there by translational yield of spliced mRNAs in mammalian cells. Proteins of the EJC are also involved in the localization of specific transcripts in D.
melanogaster embryos. Hence, the EJC represents a key modulator of post-transcriptional mRNA function and metabolism in higher eukaryotes and as an indispensable marker for gene expression quality control. Understanding the molecular processes that determine the assembly of the EJC are important to understand EJC-dependent post-transcriptional steps of gene expression.
Using mutation analysis of EJC components and a newly developed functional assay (tethering assay) for NMD proteins, we have previously identified functionally critical interactions between core EJC proteins and subsequently recruited NMD factors (Gehring et al., 2003). Specifically, we have identified an NMD-activating complex containing UPF3b and Y14 and described for the first time an essential function of Y14 in human NMD. Moreover, we showed that different EJC components specify distinct routes of NMD with differential cofactor requirements (Gehring et al., 2005). Thus, NMD factors can be grouped into different functional classes. Surprisingly, only a subset of NMD proteins requires high levels of UPF2, a factor previously thought to be one of the key proteins in NMD.
More recently, we have established an experimental system to investigate the splicing-dependent deposition of EJC components onto an RNA and demonstrate that the EJC is assembled along a defined hierarchical pathway that guarantees the proper positioning and stable binding of the EJC on the (pre-)mRNA substrate. Our data define a stable minimal pre-EJC core consisting of eIF4A3 and MAGOH-Y14, which serves as a binding platform for EJC binding factors like BTZ and UPF3b that link the EJC to functional downstream effectors (Gehring et al., 2009a). We have also identified the cytoplasmic protein PYM as an EJC disassembly factor, and defined its mechanism of function using in vitro splicing reactions. Within cells, translation-independent disassembly of EJCs by PYM appears to be prevented by its stable association with ribosomes, which restricts the activity of PYM to mRNAs that are translated. We also show that the overexpression of PYM inhibits NMD, indicating that PYM antagonizes EJC functions in living cells (Gehring et al., 2009b).
Publications
Dang, Y., Low, W.-K., Gehring, N.H., Dietz, H.C., Romo, D., Liu, J.O. (2009) Pateamine A inhibits nonsense-mediated mRNA decay by targeting eIF4AIII independent of translation initiation. J. Biol. Chem., in press
Gehring, N.H., Lamprinaki, S., Hentze, M.W., Kulozik, A.E. (2009) The hierarchy of exon-junction complex assembly by the spliceosome explains key features of mammalian nonsense-mediated mRNA decay. PLoS Biology 7(5): e1000120. doi:10.1371/journal.pbio.1000120
Clerici, M., Mourão, A., Gutsche, I., Gehring, N. H., Hentze, M. W.,Kulozik, A. E., Kadlec, J., Sattler, M., Cusack, S. (2009) Unusual bipartite mode of interaction between the nonsense mediated decay factors UPF1 and UPF2, EMBO J., 28, 2293-2306
Gehring, N.H., Lamprinaki, S., Kulozik, A.E., Hentze, M.W. (2009) Disassembly of exon junction complexes by PYM. Cell, 137, 536-48
Guenther, U.-P., Handoko, L., Laggerbauer, B., Jablonka, S., Chari, A., Alzheimer, M., Ohmer, J., Plöttner, O., Gehring, N., Sickmann, A., von Au, K., Schuelke, M., Fischer, U. (2009) IGHMBP2 is a ribosome-associated helicase inactive in the neuromuscular disorder distal SMA type 1. (DSMA1). Hum Mol Genet. 18, 1288-300
Gehring, N.H., Hentze, M.W., Kulozik, A.E. (2008) Tethering assays to investigate nonsense-mediated mRNA decay activating proteins. Methods Enzymol., 448, 467-82
Abrahamyan, L., Ajamian, L., Milev, M., Kulozik, A.E., Gehring, N.H., Mouland, A.J. (2008) Separable functions for Upf1 in nonsense-mediated and HIV-1 mRNA decay. RNA, 14, 914-27
Ivanov, P., Gehring, N.H., Hentze, M.W., Kulozik, A.E. (2008) 3’ determinants of premature termination codons: interplay between UPF1, the release factors, EJC and the 3’UTR. EMBO J., 27, 736-47
Viegas, M.H., Gehring, N.H., Breit, S., Hentze, M.W., Kulozik, A.E. (2007) The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the nonsense mediated decay pathway. Nucl. Acids Res., 35, 4542-51
Danckwardt, S., Kaufmann, I., Gentzel, M., Foerstner, K.U., Gantzert, A.S., Gehring, N.H., Neu-Yilik, G., Bork, P., Wilm, M., Keller, W., Hentze, M.W., Kulozik, A.E. (2007) Splicing factors stimulate polyadenylation via USEs at non-canonical 3' end formation signals. EMBO J., 11, 2658-69
Boelz, S., Neu-Yilik, G., Gehring, N.H., Hentze, M.W., Kulozik, A.E. (2006) A chemiluminescense-based reporter system to monitor nonsense-mediated mRNA decay. Biochem. Biophys. Res. Commun., 349, 186-191.
Stockklausner, C., Breit, S., Neu-Yilik, G., Echner, N., Hentze, M.W., Kulozik, A.E., Gehring, N.H. (2006) The uORF-containing thrombopoietin mRNA escapes nonsense-mediated mRNA decay. Nucl. Acids Res., 34, 2355-63.
Wahle, E., Gehring, N.H. (2006) Polyadenylation. In "Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine"; Ganten D, Ruckpaul K (Eds.). Review
Kunz, J.B., Neu-Yilik, G., Hentze, M.W., Kulozik, A.E., Gehring, N.H. (2006) Functions of hUpf3a and hUpf3b in nonsense mediated decay and translation. RNA, 12, 1015-22.
Holbrook, J., Neu-Yilik, G., Gehring, N.H., Hentze, M.W., Kulozik, A.E. (2006) Internal ribosome entry site mediated translation initiation triggers nonsense mediated decay. EMBO Rep., 7, 722-6.
Danckwardt, S., Hartmann, K., Gehring, N.H., Hentze, M.W., Kulozik, A.E. (2006) 3'end processing of the prothrombin mRNA in thrombophilia. Acta Haematologica, 115, 192-7. Review
Gehring, N.H., Kunz, J.B., Neu-Yilik, G., Breit, S., Viegas, M.H., Hentze, M.W., Kulozik, A.E. (2005) Exon-junction complex components specify distinct routes of nonsense-mediated mRNA decay with differential cofactor requirements. Mol. Cell, 20, 65-75.
Monshausen, M., Gehring, N.H., Kosik, K.S. (2004) The mammalian RNA-binding protein Staufen2 links nuclear and cytoplasmic RNA processing pathways in neurons. Neuromolecular Med., 6, 127-44.
Danckwardt, S., Gehring, N.H., Neu-Yilik, G., Hundsdoerfer, P., Pforsich, M., Frede, U., Hentze, M.W., Kulozik, A.E. (2004) The prothrombin 3'end formation signal reveals a unique architecture that is sensitive to thrombophilic gain-of-function mutations. Blood, 104, 428-35.
Neu-Yilik, G., Gehring, N.H., Hentze, M.W., Kulozik, A.E. (2004) Nonsense-mediated mRNA decay: from vacuum cleaner to Swiss army knife. Genome Biol., 5, 218. Review.
Baron-Benhamou, J., Gehring, N.H., Kulozik, A.E., Hentze, M.W. (2004) Using the lambdaN peptide to tether proteins to RNAs. Methods Mol. Biol., 257, 135-54.
Gehring, N.H., Neu-Yilik, G., Schell, T., Hentze, M.W., Kulozik, A.E. (2003) Y14 and hUpf3b form an NMD-activating complex. Mol. Cell, 11, 939-49.
Brocke, K.S., Neu-Yilik, G., Gehring, N.H., Hentze, M.W., Kulozik, A.E. (2002) The human intronless melanocortin 4-receptor gene is NMD insensitive. Hum. Mol. Genet., 11, 331-5.
Gehring, N.H., Frede, U., Neu-Yilik, G., Hundsdoerfer, P., Vetter, B., Hentze, M.W., Kulozik, A.E. (2001) Increased efficiency of mRNA 3' end formation: a new genetic mechanism contributing to hereditary thrombophilia. Nature Genet., 28, 389-92.
Lewitzky, M., Kardinal, C., Gehring, N.H., Schmidt, E.K., Konkol, B., Eulitz, M., Birchmeier, W., Schaeper, U., Feller, S.M. (2001) The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif. Oncogene, 20, 1052-62.
Neu-Yilik, G., Gehring, N.H., Thermann, R., Frede, U., Hentze, M.W., Kulozik, A.E. (2001) Splicing and 3' end formation in the definition of nonsense-mediated decay-competent human beta-globin mRNPs. EMBO J., 20, 532-40.
Schaeper, U., Gehring, N.H., Fuchs, K.P., Sachs, M., Kempkes, B., Birchmeier, W. (2000) Coupling of Gab1 to c-Met, Grb2, and Shp2 mediates biological responses. J. Cell Biol., 149, 1419-32.
Gehring, N.H., Hentze, M.W., Pantopoulos, K. (1999) Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress. J. Biol. Chem., 274, 6219-25.