Direct sequencing of totalSaccharomyces cerevisiaetRNAs by LC–MS/MS

Abstract

Among RNAs, transfer RNAs (tRNAs) contain the widest variety of abundant posttranscriptional 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 sequenceSaccharomyces cerevisiaetRNAs 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.