SNORNA-GUIDED RIBOSE METHYLATION OF RIBOSOMAL-RNA - STRUCTURAL FEATURES OF THE GUIDE RNA DUPLEX INFLUENCING THE EXTENT OF THE REACTION

Eukaryotic rRNAs contain a large number of ribose-methylated nucleotid es of elusive function which are confined to the universally conserved rRNA domains. Ribose methylation of these nucleotides is directed by a large family of small trans-acting guide RNAs, called box CID antise nse snoRNAs, Each snoRNA targets precisely one of the nucleotides to b e methylated within the pre-rRNA sequence, through transient formation of a 10-21 bp regular RNA duplex around the modification site. In thi s study we have analyzed how different features of the double-stranded RNA guide structure affect the extent of site-specific ribose methyla tion, by co-expressing an appropriate RNA substrate and its cognate ta ilored snoRNA guide in transfected mouse cells. We show that an increa sed GC content of the duplex can make up for the inhibitory effects of a helix truncation or for the presence of helix irregularities such a s a mismatched pair or a bulge nucleotide. However, some helix irregul arities dramatically inhibit the reaction and are not offset by furthe r stabilization of the duplex. Overall, the RNA duplex tolerates a muc h larger degree of irregularity than anticipated, even in the immediat e vicinity of the methylation site, which offers new prospects in the search for additional snoRNA guides. Accordingly, a few snoRNA-like se quences of uncertain status detected in the yeast Saccharomyces cerevi siae genome now appear as likely bona fide ribose methylation guides.