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 (m(3)C) modifications. In mammals, the number of tRNAs containing m(3)C modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing m(3)C formation in nuclear-encoded tRNAs have been identified, the proteins responsible for m(3)C modification in mt-tRNAs are unknown. Here, we show that m(3)C 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 m(3)C. We demonstrate that human cells deficient in METTL8 exhibit loss of m(3)C modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m(3)C 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 m(3)C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m(3)C formation in mt-tRNAs and uncover a potential function for m(3)C modification in mitochondrial tRNA structure.