TRMT6/61A-mediated m ¹ A 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 m ¹ A ( N 1-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 m ¹ 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 tRNA ^iMet is selectively and rapidly degraded by the exoribonuclease XRN2, reducing global protein synthesis and activating ATF4 expression. Furthermore, m ¹ A ₅₈ 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 tRNA ^iMet levels and reverses growth defects, identifying the XRN2-mediated surveillance of tRNA ^iMet as a primary driver of the cellular pathology. Collectively, our results uncover a pivotal role for TRMT6/61A-dependent m ¹ A in human tRNA maturation and define the molecular checkpoints essential for translational homeostasis.