m1A58 governs two steps of human initiator-methionine tRNA metabolism: maturation and XRN2-mediated decay

N{superscript 1}-methyladenosine at position 58 (m{superscript 1}A58), installed by the TRMT6/TRMT61A complex, is a highly conserved structural modification implicated in tRNA stability and human disease. In yeast, loss of m{superscript 1}A58 disrupts initiator methionine tRNA (tRNA) metabolism through defects in precursor processing and rapid tRNA decay, but whether these mechanisms operate in human cells has remained unclear. Here, using acute degron-mediated depletion of TRMT6 in human HCT116 cells combined with precursor-resolved tRNA sequencing, we show that m{superscript 1}A58 controls two distinct steps of tRNA metabolism. TRMT6 depletion selectively reduces mature tRNA while causing accumulation of precursor species retaining both 5'-leader and 3'-trailer sequences, consistent with impaired maturation. In parallel, the nuclear 5'[->]3' exonuclease XRN2 selectively degrades the residual mature hypomodified tRNA pool without affecting precursor accumulation. Loss of mature tRNA activates the integrated stress response and impairs proliferation, which is restored by XRN2 co-depletion. Together, our findings identify m{superscript 1}A58 as a coordinator of human initiator-tRNA maturation and surveillance, establishing a two-step pathway that maintains tRNA homeostasis in human cells.