TRMT6/61A-mediated m1A methylation facilitates human pre-tRNA maturation and prevents surveillance by XRN2

Transfer RNAs (tRNAs) are dynamically regulated by RNA modifications. The conserved TRMT6/61A catalyzes m1A (N1-methyladenosine) deposition at position 58. While TRMT6/61A dysregulation is linked to human diseases, its downstream processing consequences and molecular surveillance mechanisms remain unclear. Here we demonstrate that TRMT6/61A installs m1 A on precursor tRNAs prior to processing. Utilizing a dTAG rapid depletion system, we show that acute loss of TRMT6/61A swiftly reprograms the human tRNAome. Although elongator tRNA fluctuations are buffered by isodecoder redundancy, hypomethylated tRNAiMet is selectively and rapidly degraded by the exoribonuclease XRN2, reducing global protein synthesis and activating ATF4 expression. Furthermore, m1A58 is a prerequisite for tRNA end processing; its absence leads to the aberrant accumulation of unprocessed pre-tRNAs and disrupted tRNA-derived fragment (tRF) populations. Mechanistically, TRMT6/61A facilitates in vitro RNase Z cleavage, likely by promoting proper pre-tRNA folding. Lastly, XRN2 inhibition rescues tRNAiMet levels and reverses growth defects, identifying the XRN2-mediated surveillance of tRNAiMet as a primary driver of the cellular pathology. Collectively, our results uncover a pivotal role for TRMT6/61A-dependent m1A in human tRNA maturation and define the molecular checkpoints essential for translational homeostasis.