The RNA methyltransferase METTL8 installs m³C₃₂ in mitochondrial tRNAs^Thr/Ser(UCN) to optimise tRNA structure and mitochondrial translation.

Abstract

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 m³C₃₂ in the human mitochondrial (mt-)tRNA^Thr and mt-tRNA^Ser(UCN). 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 U₃₄G₃₅ and t⁶A₃₇/(ms²)i⁶A₃₇, present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C₃₂. Several lines of evidence demonstrate the influence of U₃₄, G₃₅, and the m³C₃₂ and t⁶A₃₇/(ms²)i⁶A₃₇ modifications in mt-tRNA^Thr/Ser(UCN) on the structure of these mt-tRNAs. Although mt-tRNA^Thr/Ser(UCN) lacking METTL8-mediated m³C₃₂ 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 m³C₃₂ within mt-tRNAs.