A novel platform of RNA 2′-O-methylation high-throughput and site-specific quantification tools revealed its broad distribution

Ribose 2′-O-methylation is involved in critical biological processes, but its biological functions and significance remain largely unknown due to the lack of accurate and efficient identification tools. To overcome this gap, we established an accurate high-throughput single-base Nm site screening method, NJU-seq (Nm-Judged-Universally sequencing), and a site-specific 2′-O-methylation quantification tool, Nm-VAQ (RNase H-based Nm Validation and Absolute Quantification). In this study, we identified several new Nm sites on HeLa rRNA and thousands of new Nm sites on mRNA of human and mouse cell lines, and 13 of them were selected and further validated. The 2′-O-methylation ratios of all validated Nm sites on mRNA were lower than 20%, which was out of the detection abilities of previous methods. Our study suggested that the function and regulation of Nm sites are more complicated than previous studies revealed and worth further mechanistic investigation.