TARGETED RIBOSE METHYLATION OF RNA IN-VIVO DIRECTED BY TAILORED ANTISENSE RNA GUIDES

EUKARYOTIC ribosomal RNAs are post-transcriptionally modified by methy lation at the ribose sugar of specific nucleotides(1). This takes plac e in the nucleolus and involves a family of small nucleolar RNAs (snoR NAs) with long regions (10-21 nucleotides) complementary to rRNA seque nces spanning the methylation site(2-4)-a complementary snoRNA is requ ired for methylation at a specific site(5). Here we show that altering the sequence of the snoRNA is sufficient to change the specificity of methylation. Mammalian cells transfected with a snoRNA engineered to be complementary to an arbitrary rRNA sequence direct the methylation of the predicted nucleotide in that sequence. We have further identifi ed structural features, both of the guide and substrate RNA, required for methylation and have used these to design an exogenous transcript, devoid of rRNA sequence, that is site-specifically methylated when co -expressed with an appropriate guide snoRNA. Endogenous non-ribosomal RNA can thus be targeted, possibly providing a highly selective tool f or the alteration of gene expression at the post-transcriptional level .