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.
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.