Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNA mutation.

The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNA The primary defect in this mutation is an alteration in tRNA aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNA 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene () in cytoplasmic hybrid (cybrid) cells carrying the m.12201T>C mutation reverses mitochondrial dysfunctions. Using molecular dynamics simulations, we demonstrate that the m.12201T>C mutation perturbs the tRNA structure and function, supported by decreased melting temperature, conformational changes, and instability of mutated tRNA. We show that the m.12201T>C mutation-induced alteration of aminoacylation tRNA causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNA from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, overexpression increased the steady-state levels of tRNA and of noncognate tRNAs, including tRNA, tRNA, tRNA, tRNA, tRNA, and tRNA, in cells bearing the m.12201T>C mutation. This improved tRNA metabolism elevated the efficiency of mitochondrial translation, activities of oxidative phosphorylation complexes, and respiration capacity. Furthermore, overexpression markedly increased mitochondrial ATP levels and membrane potential and reduced production of reactive oxygen species in cells carrying the m.12201T>C mutation. These results indicate that overexpression corrects the mitochondrial dysfunction caused by the tRNA mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.