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Mechanistic insights into mitochondrial tRNA^Ala 3'-end metabolism deficiency.

ABSTRACT: Mitochondrial tRNA 3'-end metabolism is critical for the formation of functional tRNAs. Deficient mitochondrial tRNA 3'-end metabolism is linked to an array of human diseases, including optic neuropathy, but their pathophysiology remains poorly understood. In this report, we investigated the molecular mechanism underlying the Leber's hereditary optic neuropathy (LHON)-associated tRNA^Ala 5587A>G mutation, which changes a highly conserved adenosine at position 73 (A73) to guanine (G73) on the 3'-end of the tRNA acceptor stem. The m.5587A>G mutation was identified in three Han Chinese families with suggested maternal inheritance of LHON. We hypothesized that the m.5587A>G mutation altered tRNA^Ala 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNA^Ala precursors by RNase Z and inhibited the addition of CCA by tRNA nucleotidyltransferase (TRNT1). Northern blot analysis revealed that the m.5587A>G mutation perturbed tRNA^Ala aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNA^Ala in these cells. The impact of m.5587A>G mutation on tRNA^Ala function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNA^Ala metabolism impaired mitochondrial translation, perturbed assembly and activity of oxidative phosphorylation complexes, diminished ATP production and membrane potential, and increased production of reactive oxygen species. These pleiotropic defects elevated apoptotic cell death and promoted mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual impairment. Our findings may provide new insights into the pathophysiology of LHON arising from mitochondrial tRNA 3'-end metabolism deficiency.

SUBMITTER: Ji Y

PROVIDER: S-EPMC8212662 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:Biallelic pathogenic variants in PLPBP (formerly called PROSC) have recently been shown to cause a novel form of vitamin B6-dependent epilepsy, the pathophysiological basis of which is poorly understood. When left untreated, the disease can progress to status epilepticus and death in infancy. Here we present 12 previously undescribed patients and six novel pathogenic variants in PLPBP. Suspected clinical diagnoses prior to identification of PLPBP variants included mitochondrial encephalopathy (two patients), folinic acid-responsive epilepsy (one patient) and a movement disorder compatible with AADC deficiency (one patient). The encoded protein, PLPHP is believed to be crucial for B6 homeostasis. We modelled the pathogenicity of the variants and developed a clinical severity scoring system. The most severe phenotypes were associated with variants leading to loss of function of PLPBP or significantly affecting protein stability/PLP-binding. To explore the pathophysiology of this disease further, we developed the first zebrafish model of PLPHP deficiency using CRISPR/Cas9. Our model recapitulates the disease, with plpbp-/- larvae showing behavioural, biochemical, and electrophysiological signs of seizure activity by 10 days post-fertilization and early death by 16 days post-fertilization. Treatment with pyridoxine significantly improved the epileptic phenotype and extended lifespan in plpbp-/- animals. Larvae had disruptions in amino acid metabolism as well as GABA and catecholamine biosynthesis, indicating impairment of PLP-dependent enzymatic activities. Using mass spectrometry, we observed significant B6 vitamer level changes in plpbp-/- zebrafish, patient fibroblasts and PLPHP-deficient HEK293 cells. Additional studies in human cells and yeast provide the first empirical evidence that PLPHP is localized in mitochondria and may play a role in mitochondrial metabolism. These models provide new insights into disease mechanisms and can serve as a platform for drug discovery.

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Mechanistic insights into mitochondrial tRNA^Ala 3'-end metabolism deficiency.

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Dataset Information

Mechanistic insights into mitochondrial tRNAAla 3'-end metabolism deficiency.

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OmicsDI is part of the ELIXIR infrastructure

Tweets

Mechanistic insights into mitochondrial tRNA^Ala 3'-end metabolism deficiency.