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 unanno-tated 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.

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

Anselmi F.;Neri F.;Maldotti M.;Rapelli S.;Parlato C.;Basile G.;Oliviero S.
2017-01-01

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 unanno-tated 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.
2017
45
3
1433
1441
https://academic.oup.com/nar/article/45/3/1433/2972190
Animals; Conserved Sequence; Embryonic Stem Cells; Gene Expression Profiling; HeLa Cells; High-Throughput Nucleotide Sequencing; Humans; Methylation; Mice; Nucleic Acid Conformation; RNA Processing, Post-Transcriptional; RNA, Ribosomal; RNA, Ribosomal, 18S; RNA, Ribosomal, 28S; RNA, Small Nucleolar; Sequence Analysis, RNA; Transcriptome
Incarnato D.; Anselmi F.; Morandi E.; Neri F.; Maldotti M.; Rapelli S.; Parlato C.; Basile G.; Oliviero S.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1764075
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