A mass spectrometry-based method for comprehensive quantitative determination of post-transcriptional RNA modifications: the complete chemical structure of Schizosaccharomyces pombe ribosomal RNAs.

Masato Taoka,Yuko Nobe,Shunpei Masaki,Toshiaki Isobe,Masayuki Hori,Yoshio Yamauchi,Nobuhiro Takahashi,Hiroshi Nakayama,Aiko Takeuchi

doi:10.1093/nar/gkv560

Abstract

We present a liquid chromatography–mass spectrometry (LC-MS)-based method for comprehensive quantitative identification of post-transcriptional modifications (PTMs) of RNA. We incorporated an in vitro-transcribed, heavy isotope-labeled reference RNA into a sample RNA solution, digested the mixture with a number of RNases and detected the post-transcriptionally modified oligonucleotides quantitatively based on shifts in retention time and the MS signal in subsequent LC-MS. This allowed the determination and quantitation of all PTMs in Schizosaccharomyces pombe ribosomal (r)RNAs and generated the first complete PTM maps of eukaryotic rRNAs at single-nucleotide resolution. There were 122 modified sites, most of which appear to locate at the interface of ribosomal subunits where translation takes place. We also identified PTMs at specific locations in rRNAs that were altered in response to growth conditions of yeast cells, suggesting that the cells coordinately regulate the modification levels of RNA.

Highlights

To date, >100 different types of post-transcriptional modifications (PTMs) have been found in various types of cellular RNAs, including transfer (t)RNA, rRNA, small nuclear RNA, small nucleolarRNA and messenger (m)RNA [1]
Those analytical strategies are typically based on RNase mapping, in which PTMs are identified by the mass shifts of oligonucleotides within an RNase-digested sample relative to the theoretical masses predicted from the gene or cDNA sequence
SILNAS is based on the fact that (i) an in vitro-transcribed RNA does not carry PTMs and (ii) RNA PTMs generally cause a shift in the retention time (Rt) and MS signal in liquid chromatography–mass spectrometry (LC-MS)

Summary

Introduction

To date, >100 different types of PTMs have been found in various types of cellular RNAs, including transfer (t)RNA, rRNA, small nuclear RNA, small nucleolar (sno)RNA and messenger (m)RNA [1]. SILNAS provides quantitative information about PTMs so that we can estimate the stoichiometry of the PTM at each modification site by comparing the signal heights of the unmodified light and heavy RNA fragments (Figure 1).

Results

Conclusion