Sun, J.; Wu, J.; Yuan, Y.; Fan, L.; Chua, W. L. P.; Ling, Y. H. S.; Balamkundu, S.; Dwijapriya, D.; Suen Suen, H. C.; de Crecy-Lagard, V.; Dziergowska, A.; Dedon, P.

Transfer RNA (tRNA) modifications have emerged as critical posttranscriptional 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 5,746 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 demonstrates its utility for biomarker and drug discovery and development.