Multifaceted roles of t⁶A biogenesis in efficiency and fidelity of mitochondrial gene expression

N-6-Threonylcarbamoyladenosine at A37 (t(6)A37) of ANN-decoding transfer RNAs (tRNAs) is a universal modification whose functions have been well documented in bacteria and lower eukaryotes; however, its role in organellar translation is not completely understood. In this study, we deleted the mitochondrial t(6)A37-modifying enzyme OSGEPL1 in HEK293T cells. OSGEPL1 is dispensable for cell viability. t(6)A37 hypomodification selectively stimulated N-1-methyladenosine at A9 (m(1)A9) and N-2-methylguanosine at G10 (m(2)G10) modifications and caused a substantial reduction in the aminoacylation of mitochondrial tRNA(Thr) and tRNA(Lys), resulting in impaired translation efficiency. Multiple types of amino acid misincorporation due to the misreading of near-cognate codons by t(6)A37-unmodified tRNAs were detected, indicating a triggered translational infidelity. Accordingly, the alterations in mitochondrial structure, function, and the activated mitochondrial unfolded protein response were observed. Mitochondrial function was efficiently restored by wild-type, but not by tRNA-binding-defective OSGEPL1. Lastly, in Osgepl1 deletion mice, disruption to mitochondrial translation was evident but resulted in no observable deficiency under physiological conditions in heart, which displays the highest Osgepl1 expression. Taken together, our data delineate the multifaceted roles of mitochondrial t(6)A37 modification in translation efficiency and quality control in mitochondria.