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Defects in mitochondrial RNA metabolism have been linked to sensorineural deafness that often occurs as a consequence of damaged or deficient inner ear hair cells. In this report, we investigated the molecular mechanism underlying a deafnessassociated tRNAPhe 593T > C mutation that changed a highly conserved uracil to cytosine at position 17 of the DHU-loop. The m.593T > C mutation altered tRNAPhe structure and function, including increased melting temperature, resistance to S1 nuclease-mediated digestion, and conformational changes. The aberrant tRNA metabolism impaired mitochondrial translation, which was especially pronounced by decreases in levels of ND1, ND5, CYTB, CO1, and CO3 harboring higher numbers of phenylalanine. These alterations resulted in aberrant assembly, instability, and reduced activities of respiratory chain enzyme complexes I, III, IV, and intact supercomplexes overall. Furthermore, we found that the m.593T > C mutation caused markedly diminished membrane potential, and increased the production of reactive oxygen species in the mutant cell lines carrying the m.593T > C mutation. These mitochondrial dysfunctions led to the mitochondrial dynamic imbalance via increasing fission with abnormal mitochondrial morphology. Excessive fission impaired the process of autophagy including the initiation phase, formation, and maturation of the autophagosome. In particular, the m.593T > C mutation upregulated the PARKIN-dependent mitophagy pathway. These alterations promoted an intrinsic apoptotic process for the removal of damaged cells. Our findings provide critical insights into the pathophysiology of maternally inherited deafness arising from tRNA mutation-induced defects in mitochondrial and cellular integrity. [ABSTRACT FROM AUTHOR]

Mammalian mitochondrial tRNA (mt-tRNA) plays a central role in the synthesis of the 13 subunits of the oxidative phosphorylation complex system (OXPHOS). However, many aspects of the context-dependent expression of mt-tRNAs in mammals remain unknown. To investigate the tissue-specific effects of mt-tRNAs, we performed a comprehensive analysis of mitochondrial tRNA expression across five mice tissues (brain, heart, liver, skeletal muscle, and kidney) using Northern blot analysis. Striking differences in the tissue-specific expression of 22 mt-tRNAs were observed, in some cases differing by as much as tenfold from lowest to highest expression levels among these five tissues. Overall, the heart exhibited the highest levels of mt-tRNAs, while the liver displayed markedly lower levels. Variations in the levels of mt-tRNAs showed significant correlations with total mitochondrial DNA (mtDNA) contents in these tissues. However, there were no significant differences observed in the 2-thiouridylation levels of tRNALys, tRNAGlu, and tRNAGln among these tissues. A wide range of aminoacylation levels for 15 mt-tRNAs occurred among these five tissues, with skeletal muscle and kidneys most notably displaying the highest and lowest tRNA aminoacylation levels, respectively. Among these tissues, there was a negative correlation between variations in mt-tRNA aminoacylation levels and corresponding variations in mitochondrial tRNA synthetases (mt-aaRS) expression levels. Furthermore, the variable levels of OXPHOS subunits, as encoded by mtDNA or nuclear genes, may reflect differences in relative functional emphasis for mitochondria in each tissue. Our findings provide new insight into the mechanism of mt-tRNA tissue-specific effects on oxidative phosphorylation. [ABSTRACT FROM AUTHOR]

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 tRNAAla 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 tRNAAla 3'-end metabolism and mitochondrial function. In vitro processing experiments showed that the m.5587A>G mutation impaired the 3'-end processing of tRNAAla 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 tRNAAla aminoacylation, as evidenced by decreased efficiency of aminoacylation and faster electrophoretic mobility of mutated tRNAAla in these cells. The impact of m.5587A>G mutation on tRNAAla function was further supported by increased melting temperature, conformational changes, and reduced levels of this tRNA. Failures in tRNAAla 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. [ABSTRACT FROM AUTHOR]

Mitochondriamaintain a distinct pool of ribosomal machinery, including tRNAs and tRNAs activating enzymes, such as mitochondrial tyrosyl-tRNAsynthetase (YARS2). Mutations in YARS2, which typically lead to the impairment of mitochondrial protein synthesis, have been linked to an array of human diseases including optic neuropathy. However, the lack of YARS2mutation animal model makes us difficult to elucidate the pathophysiology underlying YARS2 deficiency. To explore this system, we generated YARS2 knockout (KO) HeLa cells and zebrafish using CRISPR/Cas9 technology. We observed the aberrant tRNATyr aminoacylation overall and reductions in the levels in mitochondrion- and nucleus-encoding subunits of oxidative phosphorylation system(OXPHOS), which were especially pronounced effects in the subunits of complex I and complex IV. These deficiencies manifested the decreased levels of intact supercomplexes overall. Immunoprecipitationassays showedthatYARS2 boundto specific subunits of complex I and complex IV, suggesting the posttranslational stabilization of OXPHOS. Furthermore, YARS2 ablation caused defects in the stability and activities of OXPHOS complexes. These biochemical defects could be rescued by the overexpression of YARS2cDNAin the YARS2KO cells. In zebrafish, the yars2KO larva conferred deficient COX activities in the retina, abnormal mitochondrial morphology, and numbers in the photoreceptor and retinal ganglion cells. The zebrafish further exhibited the retinal defects affecting both rods and cones. Vision defects in yars2KO zebrafish recapitulated the clinical phenotypes in the optic neuropathy patients carrying the YARS2 mutations. Our findings highlighted the critical role of YARS2 in the stability and activity of OXPHOS and its pathological consequence in vision impairments. [ABSTRACT FROM AUTHOR]

Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (p°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations. [ABSTRACT FROM AUTHOR]

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 1220- 1T>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. [ABSTRACT FROM AUTHOR]

Nuclear modifier genes have been proposed to modify the phenotypic expression of mitochondrial DNA mutations. Using a targeted exome-sequencing approach, here we found that the p.191Gly>Val mutation in mitochondrial tyrosyl-tRNA synthetase 2 (YARS2) interacts with the tRNASer(UCN) 7511A>Gmutation in causing deafness. Strikingly, members of a Chinese family bearing both the YARS2 p.191Gly>Val and m.7511A>G mutations displayed much higher penetrance of deafness than those pedigrees carrying only the m.7511A>G mutation. The m.7511A>G mutation changed the A4:U69 base-pairing to G4:U69 pairing at the aminoacyl acceptor stem of tRNASer(UCN) and perturbed tRNASer(UCN) structure and function, including an increased melting temperature, altered conformation, instability, and aberrant aminoacylation of mutant tRNA. Using lymphoblastoid cell lines derived from symptomatic and asymptomatic members of these Chinese families and control subjects, we show that cell lines harboring only the m.7511A>G or p.191Gly>Val mutation revealed relatively mild defects in tRNASer(UCN) or tRNATyr metabolism, respectively. However, cell lines harboring both m.7511A>G and p.191Gly>Val mutations displayed more severe defective aminoacylations and lower tRNASer(UCN) and tRNATyr levels, aberrant aminoacylation, and lower levels of other tRNAs, including tRNAThr, tRNALys, tRNALeu(UUR), and tRNASer(AGY), than those in the cell lines carrying only the m.7511A>G or p.191Gly>Val mutation. Furthermore, mutant cell lines harboring both m.7511A>Gand p.191Gly>Val mutations exhibited greater decreases in the levels of mitochondrial translation, respiration, and mitochondrial ATP and membrane potentials, along with increased production of reactive oxygen species. Our findings provide molecularlevel insights into the pathophysiology of maternally transmitted deafness arising from the synergy between tRNASer(UCN) and mitochondrial YARS mutations. [ABSTRACT FROM AUTHOR]

The 1555A→G mutation in mitochondrial 12S rRNA has been associated with aminoglycoside-induced and non-syndromic deafness in many individuals worldwide. Mitochondrial genetic modifiers are proposed to influence the phenotypic expression of m.1555A→G mutation. Here, we report that a deafness-susceptibility allele (m.4317A→G) in the tRNAIle gene modulates the phenotype expression of m.1555A→G mutation. Strikingly, a large Han Chinese pedigree carrying both m.4317A→G and m.1555A→G mutations exhibited much higher penetrance of deafness than those carrying only the m.1555A→G mutation. The m.4317A→G mutation affected a highly conserved adenine at position 59 in the T-loop of tRNAIle. We therefore hypothesized that the m.4317A→G mutation alters both structure and function of tRNAIle. Using lymphoblastoid cell lines derived from members of Chinese families (three carrying both m.1555A→G and m.4317A→G mutations, three harboring only m.1555A→G mutation, and three controls lacking these mutations), we found that the cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited more severe mitochondrial dysfunctions than those carrying only the m.1555A→G mutation. We also found that the m.4317A→G mutation perturbed the conformation, stability, and aminoacylation efficiency of tRNAIle. These m.4317A→G mutation-induced alterations in tRNAIle structure and function aggravated the defective mitochondrial translation and respiratory phenotypes associated with the m.1555A→G mutation. Furthermore, mutant cell lines bearing both m.4317A→G and m.1555A→G mutations exhibited greater reductions in the mitochondrial ATP levels and membrane potentials and increasing production of reactive oxygen species than those carrying only the m.1555A→G mutation. Our findings provide new insights into the pathophysiology of maternally inherited deafness arising from the synergy between mitochondrial 12S rRNA and tRNA mutations. [ABSTRACT FROM AUTHOR]

Defective nucleotide modifications of mitochondrial tRNAs have been associated with several human diseases, but their pathophysiology remains poorly understood. In this report, we investigated the pathogenic molecular mechanism underlying a hypertension-associated 4435A→G mutation in mitochondrial tRNAMet. The m.4435A→G mutation affected a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon, which is important for the fidelity of codon recognition and stabilization. We hypothesized that the m.4435→3Gmutation introduced anm1G37 modification of tRNAMet, altering its structure and function. Primer extension and methylation activity assays indeed confirmed that the m.4435A→Gmutation created a tRNA methyltransferase 5 (TRMT5)-catalyzed m¹G37 modification of tRNAMet. We found that this mutation altered the tRNAMet structure, indicated by an increased melting temperature and electrophoretic mobility of the mutated tRNA compared with the wildtype molecule. We demonstrated that cybrid cell lines carrying the m.4435A→G mutation exhibited significantly decreased efficiency in aminoacylation and steadystate levels of tRNAMet, as compared with those of control cybrids. The aberrant tRNAMet metabolism resulted in variable decreases in mitochondrial DNA (mtDNA)-encoded polypeptides in the mutant cybrids. Furthermore, we found that the m.4435A→G mutation caused respiratory deficiency, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in mutant cybrids. These results demonstrated that an aberrant m1G37 modification of mitochondrial tRNAMet affected the structure and function of its tRNA and consequently altered mitochondrial function. Our findings provide critical insights into the pathophysiology of maternally inherited hypertension, which is manifested by the deficient tRNA nucleotide modification. [ABSTRACT FROM AUTHOR]

Several mitochondrial tRNA mutations have been associated with hypertension, but their pathophysiology remains poorly understood. In this report, we identified a novel homoplasmic 3253T3C mutation in the mitochondrial tRNALeu(UUR) gene in a Han Chinese family with maternally inherited hypertension. The m.3253T→C mutation affected a highly conserved uridine at position 22 at the D-stem of tRNALeu(UUR), introducing a G-C base pairing (G13-C22) at the D-stem and a tertiary base pairing (C22-G46) between the D-stem and the variable loop.Wetherefore hypothesized that the m.3253T→C mutation altered both the structure and function of tRNALeu(UUR). Using cytoplasmic hybrid (cybrid) cell lines derived from this Chinese family, we demonstrated that the m.3253T→C mutation perturbed the conformation and stability of tRNALeu(UUR), as suggested by faster electrophoretic mobility of mutated tRNA relative to the wild-type molecule. Northern blot analysis revealed an ∼45% decrease in the steady-state level of tRNALeu(UUR) in the mutant cell lines carrying the m.3253T→C mutation, as compared with control cell lines. Moreover, an ∼35% reduction in aminoacylation efficiency of tRNALeu(UUR) was observed in the m.3253T→C mutant cells. These alterations in tRNALeu(UUR) metabolism impaired mitochondrial translation, especially for those polypeptides with a high proportion of Leu(UUR) codons, such as ND6. Furthermore, we demonstrated that the m.3253T→C mutation decreased the activities of mitochondrial complexes I and V, markedly diminished mitochondrial ATP levels and membrane potential, and increased the production of reactive oxygen species in the cells. In conclusion, our findings may provide new insights into the pathophysiology of maternally inherited hypertension. [ABSTRACT FROM AUTHOR]

Several mitochondrial tRNA mutations have been associated with maternally inherited diabetes and deafness. However, the pathophysiology of these tRNA mutations remains poorly understood. In this report, we identified the novel homoplasmic 14692A→G mutation in the mitochondrial tRNAGlu gene among three Han Chinese families with maternally inherited diabetes and deafness. The m.14692A→G mutation affected a highly conserved uridine at position 55 of the TψC loop of tRNAGlu. The uridine is modified to pseudouridine (ψ55), which plays an important role in the structure and function of this tRNA. Using lymphoblastoid cell lines derived from a Chinese family, we demonstrated that the m.14692A→G mutation caused loss of ψ55 modification and increased angiogenin-mediated endonucleolytic cleavage in mutant tRNAGlu. The destabilization of base-pairing (18A-ψ55) caused by the m.14692A→G mutation perturbed the conformation and stability of tRNAGlu. An approximately 65% decrease in the steady-state level of tRNAGlu was observed in mutant cells compared with control cells. A failure in tRNAGlu metabolism impaired mitochondrial translation, especially for polypeptides with a high proportion of Glutamic acid codons such as ND1, ND6, and CO2 in mutant cells. An impairment of mitochondrial translation caused defective respiratory capacity, especially reducing the activities of complexes I and IV. Furthermore, marked decreases in the levels of mitochondrial ATP and membrane potential were observed in mutant cells. These mitochondrial dysfunctions caused an increasing production of reactive oxygen species in the mutant cells. Our findings may provide new insights into the pathophysiology of maternally inherited diabetes and deafness, which is primarily manifested by the deficient nucleotide modification of mitochondrial tRNAGlu. [ABSTRACT FROM AUTHOR]

Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA. [ABSTRACT FROM AUTHOR]