Direct sequencing of totalSaccharomyces cerevisiaetRNAs by LC–MS/MS
Open Access
- 11 May 2023
- journal article
- research article
- Published by Cold Spring Harbor Laboratory in RNA
- Vol. 29 (8), 1201-1214
- https://doi.org/10.1261/rna.079656.123
Abstract
Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant post-transcriptional chemical modifications. These modifications are crucial for tRNAs to participate in protein synthesis, promoting proper tRNA structure and aminoacylation, facilitating anticodon:codon recognition, and ensuring the reading frame maintenance of the ribosome. While tRNA modifications were long thought to be stoichiometric, it is becoming increasingly apparent that these modifications can change dynamically in response to the cellular environment. The ability to broadly characterize the fluctuating tRNA modification landscape will be essential for establishing the molecular level contributions of individual sites of tRNA modification. The locations of modifications within individual tRNA sequences can be mapped using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In this approach, a single tRNA species is purified, treated with ribonucleases and the resulting single-stranded RNA products are subject to LC-MS/MS analysis. The application of LC-MS/MS to study tRNAs is limited by the necessity of analyzing one tRNA at a time because the digestion of total tRNA mixtures by commercially available ribonucleases produces many short digestion products unable to be uniquely mapped back to a single site within a tRNA. We overcame these limitations by taking advantage of the highly structured nature of tRNAs to prevent the full digestion by single-stranded RNA specific ribonucleases. Folding total tRNA prior to digestion allowed us to sequence S. cerevisiae tRNAs with up to 97% sequence coverage for individual tRNA species by LC-MS/MS. This method presents a robust avenue for directly detecting the distribution of modifications in total tRNAs.Keywords
Funding Information
- National Science Foundation
- Research Corporation for Science Advancement
- University of Michigan Chemistry Mass Spectrometry Core
- National Institutes of Health
This publication has 62 references indexed in Scilit:
- Degradation of initiator tRNA Met by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cellsNucleic Acids Research, 2013
- Characterization of Modified RNA by Top‐Down Mass SpectrometryAngewandte Chemie, 2012
- Reprogramming of tRNA modifications controls the oxidative stress response by codon-biased translation of proteinsNature Communications, 2012
- Removal of 3'‐phosphate group by bacterial alkaline phosphatase improves oligonucleotide sequence coverage of RNase digestion products analyzed by collision‐induced dissociation mass spectrometryRapid Communications in Mass Spectrometry, 2011
- A Quantitative Systems Approach Reveals Dynamic Control of tRNA Modifications during Cellular StressPLoS Genetics, 2010
- Pseudouridine at position 55 in tRNA controls the contents of other modified nucleotides for low-temperature adaptation in the extreme-thermophilic eubacterium Thermus thermophilusNucleic Acids Research, 2010
- Top-down mass spectrometry for sequencing of larger (up to 61 nt) RNA by CAD and EDDJournal of the American Society for Mass Spectrometry, 2010
- Detection of the Common RNA Nucleoside Pseudouridine in Mixtures of Oligonucleotides by Mass SpectrometryAnalytical Chemistry, 2005
- Analysis of Oligonucleotides by HPLC−Electrospray Ionization Mass SpectrometryAnalytical Chemistry, 1997
- Structure mapping of 5'-32P-labeled RNA with S1 nucleaseBiochemistry, 1978