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.