A natural non-Watson–Crick base pair in human mitochondrial tRNA(Thr) causes structural and functional susceptibility to local mutations

Six pathogenic mutations have been reported in human mitochondrial tRNA(Thr) (hmtRNA(Thr)); however, the pathogenic molecular mechanism remains unclear. Previously, we established an activity assay system for human mitochondrial threonyl-tRNA synthetase (hmThrRS). In the present study, we surveyed the structural and enzymatic effects of pathogenic mutations in hmtRNA(Thr) and then focused on m.15915 G > A (G30A) and m.15923A > G (A38G). The harmful evolutionary gain of non-Watson–Crick base pair A29/C41 caused hmtRNA(Thr) to be highly susceptible to mutations disrupting the G30–C40 base pair in various ways; for example, structural integrity maintenance, modification and aminoacylation of tRNA(Thr), and editing mischarged tRNA(Thr). A similar phenomenon was observed for hmtRNA(Trp) with an A29/C41 non-Watson–Crick base pair, but not in bovine mtRNA(Thr) with a natural G29–C41 base pair. The A38G mutation caused a severe reduction in Thr-acceptance and editing of hmThrRS. Importantly, A38 is a nucleotide determinant for the t(6)A modification at A37, which is essential for the coding properties of hmtRNA(Thr). In summary, our results revealed the crucial role of the G30–C40 base pair in maintaining the proper structure and function of hmtRNA(Thr) because of A29/C41 non-Watson–Crick base pair and explained the molecular outcome of pathogenic G30A and A38G mutations.