The discovery of the genetic code transformed the life sciences by providing an analytical framework to study living systems. However, once the role of tRNAs was established in protein synthesis, the field moved on to other areas of the RNA world.

With the mainstream application of systems-wide approaches, tRNAs have emerged as implicated in multiple regulatory networks beyond translation. These roles have been associated to the variability in tRNA sequence and base modification. Here, a novel tRNA-seq. method was devised to accurately quantify the tRNAome and outline the modification and truncation landscapes, allowing to query the concerted response of tRNAs across conditions. The method was then applied to characterise the tRNA expression for an inherited mitocondropathy caused by incorrect tRNA processing in patients.

Furthermore, recent studies have indicated that tRNA involvement beyond translation is also mediated by moonlighting functions of aminoacyl tRNA synthetases (aaRSs). For example, Tyrosyl-tRNA synthetase (YARS) was seen to translocate to the nucleus upon genotoxic stress. By applying a novel method (infSILAC), it is possible to quantitatively identify direct interactors and provide a solution to the problem of infinite ratios within a given SILAC dataset. The abundance of YARS-binding partners provided further evidence to the notion that aaRSs have functionally co-opted their tRNA binding site over evolutionary time.

Next, whether the tRNAome also underpins essential biological processes within prokaryotes was explored. By assaying the RNA-binding proteome across the E. coli lifecycle stages it was possible to identify dynamic interactors. tRNA-modifying enzymes appeared responsive across conditions, including YfiF, a predicted methyl-transferase that was subsequently validated as RNA-binding in vitro. Molecular chaperones such as DnaK and HtpG, reported to bind tRNA molecules in human cell lines, also appeared in this study. By producing the first dynamic RBPome map in bacteria, hundreds of novel candidates have been identified that reconfigure their RNA-protein interactions between growth stages.

Examining tRNA biology through multiple omics approaches and across domains, we lay the foundations for a more comprehensive understanding of the role of the tRNA molecules within cellular homeostasis and adaptation.