In summary, through the identification of two individuals with mitochondrial disease-bearing compound heterozygous variants in TRMT5, our research further expands the list of mitochondrial-tRNA-modifying enzymes vital for proper mitochondrial function. Methylation of guanine 37 in mt-tRNALeu(CUN) is diminished in fibroblasts and skeletal muscle of affected individuals and recapitulated by siRNA-induced depletion of TRMT5 in HeLa cells, consistent with mitochondrial localization of the protein. The involvement of TRMT5 in m1G formation and mitochondrial respiratory function was confirmed through the rescue of tRNALeu(CUN) m1G37 modification by complementation with wild-type TRMT5 in human fibroblasts and by validation of the pathogenic variants in a yeast model. Given that 7% of all mammalian mt-tRNA residues undergo post-transcriptional modification, and that over 30 different modified mt-tRNA positions have been so far described,46 it is anticipated that future exome sequencing analyses of individuals with clinically diagnosed mitochondrial disease will reveal further pathogenic variants within mt-tRNA modifying factors.