Single-base resolution mapping of 2′-O-methylation sites by an exoribonuclease-enriched chemical method

2′-O-methylation(Nm)is one of the most abundant RNA epigenetic modifications and plays a vital role in the post-transcriptional regulation of gene expression.Current Nm mapping approaches are normally limited to highly abundant RNAs and have significant technical hurdles in m RNAs or relatively rare non-coding RNAs(nc RNAs).Here,we developed a new method for enriching Nm sites by using RNA exoribonuclease and periodate oxidation reactivity to eliminate 2′-hydroxylated(2′-OH)nucleosides,coupled with sequencing(Nm-REP-seq).We revealed several novel classes of Nm-containing nc RNAs as well as m RNAs in humans,mice,and drosophila.We found that some novel Nm sites are present at fixed positions in different t RNAs and are potential substrates of fibrillarin(FBL)methyltransferase mediated by sno RNAs.Importantly,we discovered,for the first time,that Nm located at the 3′-end of various types of nc RNAs and fragments derived from them.Our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.