The RNA methyltransferase METTL8 installs mC in mitochondrial tRNAs to optimise tRNA structure and mitochondrial translation.

Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing mC in the human mitochondrial (mt-)tRNA and mt-tRNA. METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mt-tRNA recognition elements revealed UG and tA/(ms)iA, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C. Several lines of evidence demonstrate the influence of U, G, and the mC and tA/(ms)iA modifications in mt-tRNA on the structure of these mt-tRNAs. Although mt-tRNA lacking METTL8-mediated mC are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of mC within mt-tRNAs.