Commonality and diversity in tRNA substrate recognition in t(6)A biogenesis by eukaryotic KEOPSs

N (6)-Threonylcarbamoyladenosine (t(6)A) is a universal and pivotal tRNA modification. KEOPS in eukaryotes participates in its biogenesis, whose mutations are connected with Galloway-Mowat syndrome. However, the tRNA substrate selection mechanism by KEOPS and t(6)A modification function in mammalian cells remain unclear. Here, we confirmed that all ANN-decoding human cytoplasmic tRNAs harbor a t(6)A moiety. Using t(6)A modification systems from various eukaryotes, we proposed the possible coevolution of position 33 of initiator tRNA(Met) and modification enzymes. The role of the universal CCA end in t(6)A biogenesis varied among species. However, all KEOPSs critically depended on C32 and two base pairs in the D-stem. Knockdown of the catalytic subunit OSGEP in HEK293T cells had no effect on the steady-state abundance of cytoplasmic tRNAs but selectively inhibited tRNA(Ile) aminoacylation. Combined with in vitro aminoacylation assays, we revealed that t(6)A functions as a tRNA(Ile) isoacceptor-specific positive determinant for human cytoplasmic isoleucyl-tRNA synthetase (IARS1). t(6)A deficiency had divergent effects on decoding efficiency at ANN codons and promoted +1 frameshifting. Altogether, our results shed light on the tRNA recognition mechanism, revealing both commonality and diversity in substrate recognition by eukaryotic KEOPSs, and elucidated the critical role of t(6)A in tRNA(Ile) aminoacylation and codon decoding in human cells.