Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

Christel Enroth,Jeppe Vinther,Søren Iversen,Finn Kirpekar,Line Dahl Poulsen,Anders Albrechtsen

doi:10.1093/nar/gkz736

Christel Enroth, Jeppe Vinther + Show 4 more

Open Access

https://doi.org/10.1093/nar/gkz736

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Abstract

Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.

Highlights

It is becoming increasingly clear that RNA modifications play important roles in many cellular processes and evidence linking the proteins responsible for modification of RNA to the development of human diseases is accumulating [1]
We find that arabidopsis small ribosomal subunit (SSU) rRNA has an m7G modification at position 1581, which corresponds to the location of the SSU rRNA position modified both in human and yeast rRNA, suggesting that the SSU m7G modification is universally conserved in eukaryotes
We present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows sensitive and high throughput detection of internal m7G modifications in any type of RNA

Summary

INTRODUCTION

It is becoming increasingly clear that RNA modifications play important roles in many cellular processes and evidence linking the proteins responsible for modification of RNA to the development of human diseases is accumulating [1] This is true for methylation of guanosine position 7 (m7G), which is present at internal positions in both tRNA and rRNA. After subsequent treatment with aniline, the RNA will be cleaved at the abasic sites, thereby allowing detection by mapping of reverse transcription stops [11,21,22] This strategy forms the basis for the tRNA reduction and cleavage sequencing (TRAC-Seq method), which were used together with an m7G specific antibody based method (m7G MeRIP-seq) to confidently map the m7G modifications on mouse tRNAs [12]. M7G-MaP-seq analysis of other types of short RNAs and of E. coli and yeast mRNAs failed to identify any internally m7G modified positions

MATERIALS AND METHODS

RESULTS

Findings

DISCUSSION