Molecular basis for the activity of the Box C/D snoRNP methylation enzyme. A combined solution-state NMR, solid-sate NMR and electron microscopy approach

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Post-transcriptional modifications of both rRNA and mRNA are essential to ensure the correct functioning of several cellular machineries and regulation mechanisms. One of the most important of such post-transcriptional modifications is the methylation of the RNA at the 2’-OH position. The enzyme responsible for RNA methylation is situated in the nucleolus and is assembled around snoRNAs containing a box C/D motif. Three proteins constitute this enzyme in archea, the L7ae, which recognizes first the boxC/D RNA motif, the Nop5 protein and the fibrillarin, which carries out the methylation. Despite several years of biochemical investigation and of crystallization trials, the structure and the functional mechanisms of this fundamental cellular enzyme are still unknown. In this proposal we suggest an interdisciplinary approach to address the structural basis of the catalytic activity of the box C/D snoRNP using a combination of solution-state NMR, solid-state NMR, single particle cryo-electron microscopy, small-angle neutron scattering and molecular modelling. In addition to providing information on the mechanisms of the 2’-OH methylation reaction, this project breaks the traditional barriers among different structural biology techniques and aims to open a new way to the determination of complex structures when traditional crystallographic analysis is not applicable.