Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs.

A subset of eukaryotic tRNAs is methylated in the anticodon loop, forming 3-methylcytosine (mC) modifications. In mammals, the number of tRNAs containing mC modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing mC formation in nuclear-encoded tRNAs have been identified, the proteins responsible for mC modification in mt-tRNAs are unknown. Here, we show that mC formation in human mt-tRNAs is dependent upon the methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase, as well as with mt-tRNAs containing mC. We demonstrate that human cells deficient in METTL8 exhibit loss of mC modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of mC in METTL8-deficient cells could be rescued by re-expression of WT METTL8, but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, we found METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA, suggesting a role for mC in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for mC formation in mt-tRNAs and uncover a potential function for mC modification in mitochondrial tRNA structure.