The deafness-associated m.12201T>C mutation affects the A5-U68 base-pairing within the acceptor stem of mitochondrial tRNAHis The primary defect in this mutation is an alteration in tRNAHis aminoacylation. Here, we further investigate the molecular mechanism of the deafness-associated tRNAHis 12201T>C mutation and test whether the overexpression of the human mitochondrial histidyl-tRNA synthetase gene (HARS2) 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 tRNAHis 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 tRNAHis causes mitochondrial translational defects and respiratory deficiency. We found that the transfer of HARS2 into the cybrids carrying the m.12201T>C mutation raises the levels of aminoacylated tRNAHis from 56.3 to 75.0% but does not change the aminoacylation of other tRNAs. Strikingly, HARS2 overexpression increased the steady-state levels of tRNAHis and of noncognate tRNAs, including tRNAAla, tRNAGln, tRNAGlu, tRNALeu(UUR), tRNALys, and tRNAMet, 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, HARS2 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 HARS2 overexpression corrects the mitochondrial dysfunction caused by the tRNAHis mutation. These findings provide critical insights into the pathophysiology of mitochondrial disease and represent a step toward improved therapeutic interventions for mitochondrial disorders.

中文翻译：

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线粒体组氨酸-tRNA合成酶的过表达恢复了与耳聋相关的tRNAHis突变引起的线粒体功能障碍。

耳聋相关的m.12201T> C突变影响线粒体tRNAHis受体茎内的A5-U68碱基配对，此突变的主要缺陷是tRNAHis氨基酰化的改变。在这里，我们进一步研究与耳聋相关的tRNAHis 12201T> C突变的分子机制，并测试携带m.12201T> C突变的细胞质杂种（杂交）细胞中人线粒体组氨酸-tRNA合成酶基因（HARS2）是否过表达。逆转线粒体功能障碍。使用分子动力学模拟，我们证明了m.12201T> C突变扰动了tRNAHis的结构和功能，这由降低的解链温度，构象变化和突变的tRNA的不稳定性所支持。我们显示m.12201T> C突变诱导的氨基酰化tRNAHis的改变导致线粒体翻译缺陷和呼吸不足。我们发现，将HARS2转移到携带m.12201T> C突变的杂种中，可使氨酰化的tRNAHis的水平从56.3％提高到75.0％，但不会改变其他tRNA的氨酰化作用。令人惊讶的是，HARS2过表达提高了带有m.12201T> C突变的细胞中tRNAHis和非同源tRNA（包括tRNAAla，tRNAGln，tRNAGlu，tRNALeu（UUR），tRNALys和tRNAMet）的稳态水平。改善的tRNA代谢提高了线粒体翻译的效率，氧化磷酸化复合物的活性和呼吸能力。此外，HARS2过表达显着增加了携带m.12201T> C突变的细胞中的线粒体ATP水平和膜电位，并减少了活性氧的产生。这些结果表明，HARS2过表达纠正了由tRNAHis突变引起的线粒体功能障碍。这些发现为线粒体疾病的病理生理学提供了重要的见解，并代表着朝着改善线粒体疾病的治疗干预迈出的一步。