Development and Application of Ribose Oxidation Sequencing (RibOxi-seq)

In eukaryotes, a number of RNA sites are modified by 2’-O methylation (2’-OMe). Such editing is mostly guided by BoxC/D class small nucleolar RNAs (snoRNAs). These snoRNAs direct methylation via complementary RNA-RNA interactions. 2’-OMe has so far been shown to be present in rRNAs, tRNAs and some small RNAs and has been implicated in ribosome maturation and translational circuitries. A substantial portion of known methylated sites in rRNA lie in close proximity to ribosome functional sites such as regions around the peptidyl transferase center. It is not yet clear whether many mRNAs might possess internal 2’-OMe sites. It is therefore important to characterize 2’-OMe landscapes. We have developed a novel method for the highly accurate and transcriptome-wide detection of 2’-OMe sites. The core principle of this method is to randomly digest RNAs to expose 2’-OMe sites at the 3’-ends of digested RNA fragments. Next, an oxidation step using sodium periodate destroys all fragment-3’-ends except those that are 2’-O methylated. Only these oxidation-resistant fragments are available for linker ligation and subsequent sequencing library preparation. We have applied our new method to the study of ribose methylation in both Trypanosoma brucei and Mus musculus liver, and successfully obtained rRNA 2’-OMe profiles for both organisms. Our sequencing data strongly support experimentally validated known sites, while providing several candidates of novel sites, as well as evidence for differential methylation. In conclusion, our method is able to reliably identify 2’-OMe sites in a high-throughput manner. We are also interested in using the method to investigate potential mRNA internal sites, the targets of “orphan” snoRNAs, which have no currently known targets