An integrative platform for detection of RNA 2'-O-methylation reveals its broad distribution on mRNA.

Ribose 2'-O-methylation is involved in critical biological processes, but its biological functions and significance in mRNAs remain underexplored. We have developed NJU-seq, a sensitive method for unbiased 2'-O-methylation (Nm) profiling, and Nm-VAQ, a site-specific quantification tool. Using these tools in tandem, we identified thousands of Nm sites on mRNAs of human and mouse cell lines, of which 68 of 84 selected sites were further validated to be more than 1% 2'-O-methylated. Unlike rRNA, most mRNA Nm sites were from 1% to 30% methylated. In addition, mRNA Nm was dynamic, changing according to the circumstance. Furthermore, we show that fibrillarin is involved as a methyltransferase. By mimicking the detected Nm sites and the context sequence, the RNA fragments could be 2'-O-methylated and demonstrated higher stability but lower translation efficiency. Last, profiling of Nm sites in lung surgery samples revealed common signatures of lung cancer pathogenesis, providing potential new diagnostic markers.

Biochemical characterization of RNase R 2'-O-methylation sensitivity.

RNase R is a member of the RNA exonuclease family that digests RNA in the 3'-5' direction. Previous studies have identified RNase R from Mycoplasma genitalium (MgR) as the only RNA exonuclease that is sensitive to 2'-O-methylation (Nm) modification. However, the mechanism underlying this characteristic is not well understood. In this study, we aimed to explore the molecular mechanism of RNase R Nm sensitivity using an improved assay that can better evaluate Nm sensitivity. By comparing the sequences of five wild-type RNase R variants from Mycoplasma, we identified the importance of loop 18 in Nm sensitivity. Furthermore, we demonstrated the critical roles of L283, T278, and T279 within loop18. Our findings deepen the understanding of the molecular mechanism of why MgR is sensitive to Nm and provide a potential direction of protein engineering for applications.