tRNA modification profiling reveals epitranscriptome regulatory networks in Pseudomonas aeruginosa

Transfer RNA (tRNA) modifications have emerged as critical post-transcriptional regulators of gene expression affecting diverse biological and disease processes. While there is extensive knowledge about the enzymes installing the dozens of post-transcriptional tRNA modifications-the tRNA epitranscriptome-very little is known about how metabolic, signaling, and other networks integrate to regulate tRNA modification levels. Here, we took a comprehensive first step at understanding epitranscriptome regulatory networks by developing a high-throughput tRNA isolation and mass spectrometry-based modification profiling platform and applying it to a Pseudomonas aeruginosa transposon insertion mutant library comprising 5746 strains. Analysis of >200,000 tRNA modification data points validated the annotations of predicted tRNA modification genes, uncovered novel tRNA-modifying enzymes, and revealed tRNA modification regulatory networks in P. aeruginosa. Platform adaptation for RNA-seq library preparation would complement epitranscriptome studies, while application to human cell and mouse tissue would facilitate biomarker and drug discovery and development.