In human mitochondria, 22 species of tRNAs encoded in mitochondrial DNA (mtDNA) play critical roles in the translation of essential subunits of the respiratory chain complexes involved in oxidative phosphorylation. Mitochondrial tRNAs (mt tRNAs) are modified post-transcriptionally by nuclear-encoded tRNA-modifying enzymes. These modifications are required for the proper functioning of mt tRNAs, and lack of these modifications can cause pathological consequences. We previously reported that 5-taurinomethyluridine (τm⁵U) was not present in mt tRNA^Leu(UUR) bearing each of five pathogenic mutations associated with mitochondrial encephalopathy, lactic acidosis and stroke-like syndrome (MELAS). Lack of τm⁵U in mt tRNA was demonstrated to be a major factor to explain molecular pathogenesis of MELAS.

N⁶-threonylcarbamoyladenosine (t⁶A) is an essential tRNA modification highly conserved in all three kingdoms of life. t⁶A occurs at the position 3’ adjacent to the anticodon (position 37). t⁶A37 of tRNA plays crucial roles in aminoacylation, decoding, and translocation during protein synthesis. Among 22 mt tRNA species of human mitochondria, five mt tRNAs (mt tRNA^Thr, mt tRNA^Ile, mt tRNA^Asn, mt tRNA^Lys, and mt tRNA^Ser(AGY)) have t⁶A37. To reveal biogenesis and functional impact of this modification associated with human mitochondrial diseases, we have been studying t⁶A-modifying enzymes in human mitochondria.

In Saccharomyces cerevisiae, Sua5p and Qri7p are known to constitute a minimal enzyme system for t⁶A formation. Sua5p employs bicarbonate, L-threonine and ATP as substrates to synthesize threonylcarbamoyl-AMP (TC-AMP) which is an activated intermediate for t⁶A formation. Then, Qri7p recognizes tRNA and introduces the threonylcarbamoyl moiety of TC-AMP to N6 amino group of A37 in tRNA. Human YRDC and OSGEPL1 have been predicted to be homologs of yeast Sua5p and Qri7p, respectively. In fact, hypomodification of t⁶A was observed in mt tRNA^Ile when OSGEPL1 was knocked down in HeLa cell. We obtained both recombinant proteins and successfully reconstituted t⁶A formation of five mt tRNAs. We measured the kinetic parameters of TC-AMP formation catalyzed by human YRDC, and found that the bicarbonate concentration is the rate-limiting factor for t⁶A biogenesis, indicating that t⁶A frequency is regulated by sensing intracellular bicarbonate concentration. To date, about 200 pathogenic mutations associated with mitochondrial diseases have been reported in tRNA genes encoded on mtDNA. We are now searching for some pathogenic point mutations that affect t⁶A formation.