Dataset Information

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.

SUBMITTER: Incarnato D

PROVIDER: S-EPMC5388417 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Incarnato Danny D Anselmi Francesca F Morandi Edoardo E Neri Francesco F Maldotti Mara M Rapelli Stefania S Parlato Caterina C Basile Giulia G Oliviero Salvatore S

Nucleic acids research 20170201 3

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, an ...[more]

PMID: 28180324

Similar Datasets

Project description:The major information-carrying macromolecules in the cell, DNA, RNA, and protein, carry an additional layer of information on top of their sequence in the form of modifications of residues. The modifications provide additional functional groups and impact the structure and function of the molecules. Cellular RNA molecules contain more than 100 different modifications and are found in all domains of life and in all major classes of RNA in eukaryotic organisms. Together these modifications constitute the epitranscriptome of which two-thirds are methylations with 2’-O methylation of the ribose moiety of the nucleotide as the most abundant. Many aspects of ribose methylation are underexplored because the existing methods for their detection are laborious and can only address a few modification sites at a time. Here, we introduce RiboMeth-Seq, a high-throughput sequencing based method and applies it to yeast ribosomal RNA. We detect all of the known as well as one new methylation site and provide evidence for hypomethylation at specific residues. Furthermore we demonstrate that many methylation events are interdependent and outline the timing of modifications during ribosome biogenesis. Our results demonstrate a novel and efficient approach to understanding of the role of modifications in ribosomal RNA folding and ribosome function. Recent evidence point to changes in ribose methylation patterns in cancer ribosomes and we anticipate that RiboMeth-Seq can be applied here as well as to other diseases in which ribosomes are affected, including the heritable ribosomopathies. Yeast RNA was degraded at denaturing conditions into small fragments, long enough to be mapped by sequence alignment to the reference genome (20-40 nucleotides). The fragments were ligated to RNA oligos using a tRNA ligase that was mutated to remove its kinase activity, reverse transcribed and the cDNA used as input for Ion semiconductor sequencing. The first and last nucleotides of the inserts were recorded using the full sequence for mapping and the read-ends plotted against the sequence. 2'-O-Methylated RNA positions are protected from cleavage, and read ends from such positions are therefore underrepresented compared to the surrounding positions.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets