The structure of the box C/D enzyme reveals regulation of RNA methylation.
Lapinaite, A., Simon, B., Skjaerven, L., Rakwalska-Bange, M., Gabel, F. & Carlomagno, T.
Nature. 2013 Oct 24;502(7472):519-23. doi: 10.1038/nature12581. Epub 2013 Oct 13.
Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2'-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.
A Suite of Solid-State NMR Experiments for RNA Intranucleotide Resonance Assignment in a 21 kDa Protein-RNA Complex.
Marchanka, A., Simon, B. & Carlomagno, T.
Angew Chem Int Ed Engl. 2013 Jul 26. doi: 10.1002/anie.201304779.
Intranucleotide resonance of the 26mer box C/D RNA in complex with the L7Ae protein were assigned by solid-state NMR (ssNMR; see picture) spectroscopy. This investigation opens the way for studying RNA in large protein-RNA complexes by ssNMR spectroscopy.
Accounting for conformational variability in protein-ligand docking with NMR-guided rescoring.
Skjaerven, L., Codutti, L., Angelini, A., Grimaldi, M., Latek, D., Monecke, P., Dreyer, M.K. & Carlomagno, T.
J Am Chem Soc. 2013 Apr 17;135(15):5819-27. doi: 10.1021/ja4007468. Epub 2013 Apr8.
A key component to success in structure-based drug design is reliable information on protein-ligand interactions. Recent development in NMR techniques has accelerated this process by overcoming some of the limitations of X-ray crystallography and computational protein-ligand docking. In this work we present a new scoring protocol based on NMR-derived interligand INPHARMA NOEs to guide the selection of computationally generated docking modes. We demonstrate the performance in a range of scenarios, encompassing traditionally difficult cases such as docking to homology models and ligand dependent domain rearrangements. Ambiguities associated with sparse experimental information are lifted by searching a consensus solution based on simultaneously fitting multiple ligand pairs. This study provides a previously unexplored integration between molecular modeling and experimental data, in which interligand NOEs represent the key element in the rescoring algorithm. The presented protocol should be widely applicable for protein-ligand docking also in a different context from drug design and highlights the important role of NMR-based approaches to describe intermolecular ligand-receptor interactions.
Structural principles of RNA catalysis in a 2'-5' lariat-forming ribozyme.
Carlomagno, T., Amata, I., Codutti, L., Falb, M., Fohrer, J., Masiewicz, P. & Simon, B.
J Am Chem Soc. 2013 Mar 20;135(11):4403-11. doi: 10.1021/ja311868t. Epub 2013 Mar8.
RNA-catalyzed lariat formation is present in both eukaryotes and prokaryotes. To date we lack structural insights into the catalytic mechanism of lariat-forming ribozymes. Here, we study an artificial 2'-5' AG1 lariat-forming ribozyme that shares the sequence specificity of lariat formation with the pre-mRNA splicing reaction. Using NMR, we solve the structure of the inactive state of the ribozyme in the absence of magnesium. The reaction center 5'-guanosine appears to be part of a helix with an exceptionally widened major groove, while the lariat-forming A48 is looped out at the apex of a pseudoknot. The model of the active state built by mutational analysis, molecular modeling, and small-angle X-ray scattering suggests that A48 is recognized by a conserved adenosine, juxtaposed to the 5'-guanosine in one base-pair step distance, while the G1-N7 coordinates a magnesium ion essential for the activation of the nucleophile. Our findings offer implications for lariat formation in RNA enzymes including the mechanism of the recognition of the branch-site adenosine.
Protein-RNA Interfaces Probed by (1) H-Detected MAS Solid-State NMR Spectroscopy.
Asami, S., Rakwalska-Bange, M., Carlomagno, T. & Reif, B.
Angew Chem Int Ed Engl. 2013 Feb 18;52(8):2345-9. doi: 10.1002/anie.201208024.Epub 2013 Jan 18.
Both protonated and deuterated samples were employed in the study of the L7Ae box C/D RNA complex by (1) H-detected solid-state NMR spectroscopy. This approach yielded high-resolution spectra and was used to determine the intermolecular interface and extract structural parameters with high accuracy.
Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity.
Ballare, C., Lange, M., Lapinaite, A., Martin, G.M., Morey, L., Pascual, G., Liefke, R., Simon, B., Shi, Y., Gozani, O., Carlomagno, T., Benitah, S.A. & Di Croce, L.
Nat Struct Mol Biol. 2012 Dec;19(12):1257-65. doi: 10.1038/nsmb.2434. Epub 2012Oct 28.
Polycomb-group proteins are transcriptional repressors with essential roles in embryonic development. Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for Lys27. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of methylated histone H3 Lys36 (H3K36me), a mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. Using NMR spectroscopy, we provide structural evidence for this interaction. Furthermore, we show that Phf19 binds to a subset of PRC2 targets in mouse embryonic stem cells and that this is required for their repression and for H3K27me3 deposition. These findings show that the interaction of Phf19 with H3K36me2 and H3K36me3 is essential for PRC2 complex activity and for proper regulation of gene repression in embryonic stem cells.
Recognition of 2'-O-methylated 3'-end of piRNA by the PAZ domain of a Piwi protein.
Simon, B., Kirkpatrick, J.P., Eckhardt, S., Reuter, M., Rocha, E.A., Andrade-Navarro, M.A., Sehr, P., Pillai, R.S. & Carlomagno, T.
Structure. 2011 Feb 9;19(2):172-80. Epub 2011 Jan 20.
Piwi proteins are germline-specific Argonautes that associate with small RNAs called Piwi-interacting RNAs (piRNAs), and together with these RNAs are implicated in transposon silencing. The PAZ domain of Argonaute proteins recognizes the 3'-end of the RNA, which in the case of piRNAs is invariably modified with a 2'-O-methyl group. Here, we present the solution structure of the PAZ domain from the mouse Piwi protein, MIWI, in complex with an 8-mer piRNA mimic. The methyl group is positioned in a hydrophobic cavity made of conserved amino acids from strand beta7 and helix alpha3, where it is contacted by the side chain of methionine-382. Our structure is similar to that of Ago-PAZ, but subtle differences illustrate how the PAZ domain has evolved to accommodate distinct 3' ends from a variety of RNA substrates.