m ¹ A58 acts as a conformational checkpoint coupling human initiator tRNA maturation to translation initiation

tRNAs are characterized by extensive chemical modifications that influence tRNA fate. N ¹ -methyladenosine at position 58 (m ¹ A58) is a widespread core tRNA modification linked to physiological and pathological processes. However, how m ¹ A58 coordinate tRNA folding and processing to ensure translational efficiency in mammalian cells remains largely unknown. Using acute dTAG-mediated degradation and CRISPR-Cas9 knockout, we identified initiator methionine tRNA (tRNA ^iMet ) as selectively vulnerable to m ¹ A58 loss, lacking the isodecoder buffering observed for most other tRNA isoacceptors. NMR analysis of the tRNA ^iMet showed that m ¹ A58 stabilizes D/T-loop interactions, consistent with a maturation-competent conformation. In vitro processing assays further demonstrated that m ¹ A58 promotes RNase P-mediated 5’-leader removal and RNase Z-mediated 3’-trailer cleavage, while La/SSB protects accumulated precursors. Disrupting this checkpoint impaired the assembly of the eIF2-containing 43S pre-initiation complex and global protein synthesis, which was substantially rescued by adding m ¹ A58-modified tRNA ^iMet . Acute TRMT6 degradation elicited temporally coordinated gene-expression responses involving proteostasis, transport and signaling. Together, these findings establish m ¹ A58 as a conformational checkpoint coupling human initiator-tRNA maturation to translation initiation and stress responses.