High-throughput single-base resolution mapping of RNA 2΄-O-methylated residues

Abstract

Functional characterization of the transcriptome requires tools for the systematic investigation of RNA post-transcriptional modifications. 2΄-O-methylation (2΄-OMe) of the ribose moiety is one of the most abundant post-transcriptional modifications of RNA, although its systematic analysis is difficult due to the lack of reliable high-throughput mapping methods. We describe here a novel high-throughput approach, named 2OMe-seq, that enables fast and accurate mapping at single-base resolution, and relative quantitation, of 2΄-OMe modified residues. We compare our method to other state-of-art approaches, and show that it achieves higher sensitivity and specificity. By applying 2OMe-seq to HeLa cells, we show that it is able to recover the majority of the annotated 2΄-OMe sites on ribosomal RNA. By performing knockdown of the Fibrillarin methyltransferase in mouse embryonic stem cells (ESCs) we show the ability of 2OMe-seq to capture 2΄-O-Methylation level variations. Moreover, using 2OMe-seq data we here report the discovery of 12 previously unannotated 2΄-OMe sites across 18S and 28S rRNAs, 11 of which are conserved in both human and mouse cells, and assigned the respective snoRNAs for all sites. Our approach expands the repertoire of methods for transcriptome-wide mapping of RNA post-transcriptional modifications, and promises to provide novel insights into the role of this modification.

Evidence for rRNA 2′-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes

“…To identify potential site-specific methylation events and to quantify individual variations following FBL knockdown, we analyzed the methylation frequency of every nucleotide known to be ribose-methylated in human ribosomes (10). Several RNA-Seqbased 2′-O-Me mapping methods have been developed and used to refine the map of rRNA 2′-O-Me; however, these methods have so far not been applied to studying the dynamics of individual rRNA 2′-O-Me (9,10,28). We modified and applied our recently developed high-throughput RiboMethSeq technology (9) to human rRNA.…”

Nm-seq maps 2′-O-methylation sites in human mRNA with base precision

“…This mapping method was recently adapted to a high-throughput sequencing format (2OMe-seq) 16 . It was joined by RiboMeth-seq 17–19 , a method that leverages the resistance of 2′- O -methylated ribose to alkaline hydrolysis, leading to under-representation of these positions among read starts or ends to provide a negative readout of the methylation landscape.…”

High-throughput and site-specific identification of 2′-O-methylation sites using ribose oxidation sequencing (RibOxi-seq)

“…This makes primer extension most useful only as a confirmation tool. This method has, however, as described in RIM-seq and 2OMe-seq, recently been adapted for high-throughput detection of ribose methylation sites by combining random priming with nextgeneration sequencing (Incarnato et al 2016;Jorjani et al 2016). This study identified over 400 sites, almost 300 more than what have been curated in human rRNAs (Lestrade and Weber 2006).…”