m ¹ A58 governs two steps of human initiator-methionine tRNA metabolism: maturation and XRN2-mediated decay

N¹-methyladenosine at position 58 (m¹A ₅₈ ), installed by the TRMT6/TRMT61A complex, is a highly conserved structural modification implicated in tRNA stability and human disease. In yeast, loss of m¹A ₅₈ disrupts initiator methionine tRNA (tRNAᵢᴹᵉᵗ) 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 m¹A ₅₈ controls two distinct steps of tRNAᵢᴹᵉᵗ metabolism. TRMT6 depletion selectively reduces mature tRNAᵢᴹᵉᵗ 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 tRNAᵢᴹᵉᵗ pool without affecting precursor accumulation. Loss of mature tRNAᵢᴹᵉᵗ activates the integrated stress response and impairs proliferation, which is restored by XRN2 co-depletion. Together, our findings identify m¹A ₅₈ as a coordinator of human initiator-tRNA maturation and surveillance, establishing a two-step pathway that maintains tRNAᵢᴹᵉᵗ homeostasis in human cells.