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Mechanistic insights into mitochondrial tRNAAla 3’-end metabolism deficiency

  • Yanchun Ji
    Affiliations
    Division of Medical Genetics and Genomics, The Children’s Hospital, Zhejiang University School of Medicine and National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China

    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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  • Zhipeng Nie
    Affiliations
    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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  • Feilong Meng
    Affiliations
    Division of Medical Genetics and Genomics, The Children’s Hospital, Zhejiang University School of Medicine and National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China

    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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  • Cuifang Hu
    Affiliations
    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China

    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
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  • Hui Chen
    Affiliations
    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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  • Lihao Jin
    Affiliations
    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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  • Mengquan Chen
    Affiliations
    Department of Lab Medicine, Wenzhou Hospital of Traditional Chinese Medicine, Wenzhou, Zhejiang, China
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  • Minglian Zhang
    Affiliations
    Department of Ophthalmology, Hebei Provincial Eye Hospital, Xingtai, Hebei, China
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  • Juanjuan Zhang
    Affiliations
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
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  • Min Liang
    Affiliations
    School of Ophthalmology and Optometry, Wenzhou Medical University, Wenzhou, Zhejiang, China
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  • Meng Wang
    Affiliations
    Division of Medical Genetics and Genomics, The Children’s Hospital, Zhejiang University School of Medicine and National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China

    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
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  • Min-Xin Guan
    Correspondence
    For correspondence: Min-Xin Guan
    Affiliations
    Division of Medical Genetics and Genomics, The Children’s Hospital, Zhejiang University School of Medicine and National Clinical Research Center for Child Health, Hangzhou, Zhejiang, China

    Institute of Genetics, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China

    Center for Mitochondrial Genetics, Zhejiang Provincial Key Laboratory of Genetic & Developmental Disorders, Zhejiang Univesity, Hangzhou, Zhejiang, China

    Division of Mitochondrial Biomedicine, Joint Institute of Genetics and Genome Medicine between Zhejiang University and University of Toronto, Hangzhou, Zhejiang, China
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Open AccessPublished:May 20, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100816
      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.

      Keywords

      Abbreviations:

      A73 (adenosine at position 73), LAMP1 (lysosome-associated membrane glycoprotein 1), LHON (Leber’s hereditary optic neuropathy), mDNA (mitochondrial DNA), OCR (oxygen consumption rate), OXPHOS (oxidative phosphorylation system), PARP (poly ADP ribose polymerase), RGC (retinal ganglion cell), ROS (reactive oxidative species), TBE (Tris-borate-EDTA), TRNT1 (tRNA nucleotidyltransferase)
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      Leber’s hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees.
      ). As shown in Figure 1, the m.5587A>G mutation affected a highly conserved adenine at position 73 (A73) at accepter stem of tRNAAla. The A73 is the site for the tRNAAla 3’ end precursor processing of L-strand transcripts catalyzed by RNase Z, the CCA addition synthesized by TRNT1 and the discriminator base for its cognate aminoacyl-tRNA synthetase (
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      ,
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      ,
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      Naturally occurring mutations in human mitochondrial pre-tRNASer(UCN) can affect the transfer ribonuclease Z cleavage site, processing kinetics, and substrate secondary structure.
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      ). Therefore, it is anticipated that the A73 to G73 transition of tRNAAla caused by m.5587A>G mutation leads to pleiotropic effects on the 3’ end processing of transcript precursor, CCA addition, aminoacylation, and stability of tRNAAla. The aberrant tRNAAla metabolism may result in the impairment of mitochondrial translation, defects in oxidative phosphorylation, oxidative stress, and subsequent failure of cellular energetic processes. To investigate pathogenic mechanism of m.5587A>G mutation, we generated the cybrids by transferring mitochondria from lymphoblastoid cell lines derived from affected matrilineal relative carrying the m.5587A>G mutation and from a control subject lacking the mutation into mtDNA-less ρo206 cells (
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      ). These cybrid lines were analyzed for the effects of the m.5587A>G mutation on the tRNAAla metabolisms including the 3’ end processing of tRNA precursors, CCA addition, stability, and aminoacylation of tRNAAla. These cybrids were further assessed for the effects of m.5587A>G mutation on mitochondrial translation, oxidative phosphorylation system (OXPHOS), mitochondrial membrane potential, production of reactive oxidative species (ROS), apoptosis, and autophagy.
      Figure thumbnail gr1
      Figure 1In vitro assay for the 3’ end processing of mitochondrial tRNAAla precursors. A, mitochondrial tRNAAla precursors. Twenty nucleotides (nt) of 3’ end trailer of tRNAAla were shown, including the m.5587A>G substitution. B, in vitro 3’ end processing assays. Processing assays with mitochondrial RNase Z were undertaken in parallel for wild-type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20, 25, or 40 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis. After electrophoresis, the reaction products were visualized by staining with NA-Red (Beyotime). C, quantification of the efficiencies of tRNAAla precursors catalyzed by RNase Z. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. The error bars indicate two standard errors of the mean (SEM).

      Results

      Clinical presentation

      Three Han Chinese LHON pedigrees bearing the m.5587A>G mutation were identified among a large cohort of 1793 Chinese probands with LHON (
      • Ji Y.
      • Qiao L.
      • Liang X.
      • Zhu L.
      • Gao Y.
      • Zhang J.
      • Jia Z.
      • Wei Q.P.
      • Liu X.
      • Jiang P.
      • Guan M.X.
      Leber’s hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees.
      ). As shown in Table S1 and Fig. S1, seven of 21 matrilineal relatives exhibited variable penetrance and expressivity of optic neuropathy. In particular, the severity of visual loss ranged from profound visual loss to normal vision. The age at onset of optic neuropathy of seven affected matrilineal relatives bearing the m.5587A>G mutation ranged from 6 to 41 years, with an average of 26 years. These pedigrees exhibited different penetrance of optic neuropathy, ranging from 14.3% to 42.9%, with an average of 33.3%. There was no evidence that any of other members of these families had any other causes to account for vision loss. These matrilineal relatives showed no other clinical abnormalities, including cardiac failure, muscular diseases, visual failure, and neurological disorders. Further analysis showed that the m.5587A>G mutation was present in homoplasmy in all matrilineal relatives but not in other members of these families (data not shown).

      The aberrant 3’ end processing of tRNAAla precursors

      To examine whether the m.5587A>G mutation altered the 3’ end processing of tRNAAla precursor, we performed an in vitro processing experiment using RNase Z that was reconstituted from purified recombinant proteins ELAC2 as described previously (
      • Reinhard L.
      • Sridhara S.
      • Hallberg B.M.
      The MRPP1/MRPP2 complex is a tRNA-maturation platform in human mitochondria.
      ). As illustrated in Figure 1A, the wild-type and mutant tRNAAla precursors corresponding to mtDNA at positions 5567 to 5655 were prepared by in vitro transcription, respectively. To analyze the in vitro processing kinetics, the wild-type and mutant tRNAAla precursors were incubated with RNase Z at various time courses. The relative processing efficiencies were calculated by the ratios of cleaved pre-tRNAs at the initial phase of reaction according to the fitted curve under exponential equation (one-phase association). As shown in Figure 1B, the processing efficiencies of the mutant tRNAAla transcripts were significantly reduced, as compared with those of wild-type counterparts. As shown in Figure 1C, the processing efficiencies of mutant tRNAAla transcripts catalyzed by RNase Z were 44.2% of those in their wild-type counterparts, respectively. These results demonstrated that the m.5587A>G mutation perturbed the 3’ end processing of tRNAAla precursors.

      Impairment of 3’ CCA-adding activity of tRNAAla

      To assess whether the m.5587A>G mutation impaired the CCA-adding activity of tRNAAla, we performed an in vitro processing experiment using a CCA-adding enzyme TRNT1 (
      • Reinhard L.
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      • Hallberg B.M.
      The MRPP1/MRPP2 complex is a tRNA-maturation platform in human mitochondria.
      ,
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      tRNA 3'-amino-tailing for stable amino acid attachment.
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      Multilevel functional and structural defects induced by two pathogenic mitochondrial tRNA mutations.
      ). As showed in Figure 2A, the wild-type and mutant tRNAAla without the 3’-terminal CCA sequences corresponding to mtDNA at positions 5587 to 5655 were prepared by in vitro transcription and their capability of incorporating CCA catalyzed by the recombinant TRNT1 was then evaluated, respectively (
      • Nagaike T.
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      • Negayama F.
      • Watanabe K.
      • Ueda T.
      Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases.
      ). To analyze the in vitro processing kinetics, the wild-type and mutant tRNAAla precursors were incubated with TRNT1 at various time courses. The relative processing efficiencies were calculated by the ratios of added CCA tail at the initial phase of reaction. As shown in Figure 2, B and C, the 3’ end CCA-adding activities in the mutant transcripts were 57.9% of those in their wild-type counterparts, respectively. These results revealed that the m.5587A>G mutation inhibited the activity of CCA-adding of tRNAAla catalyzed by TRNT1.
      Figure thumbnail gr2
      Figure 2In vitro analysis for the CCA-adding activity of mitochondrial tRNAAla. A, the wild-type and mutant tRNAAla without the 3’-terminal CCA sequences were prepared by in vitro transcription and their capability of incorporating CCA was analyzed by the recombinant TRNT1. Mitochondrial tRNAAla precursors. B, in vitro analysis for the CCA-adding activity. Assays for the CCA-adding activity with CCA-adding enzyme TRNT1were carried out in parallel for wild-type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20, 25, or 40 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis. After electrophoresis, the reaction products were visualized by staining with NA-Red (Beyotime). C, quantification of the efficiencies of tRNAAla precursors catalyzed by RNase Z. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. The error bars indicate two SEM.

      Altered conformation and stability of tRNAAla

      It was anticipated that the m.5587A>G mutation caused the structural alteration and instability of tRNAAla. To experimentally test the effect of m.5877A>G mutation on the stability of tRNAAla, we examined the melting temperatures (Tm) of wild-type (A73) and mutant (G73) tRNAAla transcripts. These Tm values were determined by calculating the derivatives of the absorbance against a temperature curve (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ). As shown in Figure 3A, the Tm values for wild-type (A73) and mutant (G73) transcripts were 33.6 °C ± 0.7 °C and 39.6 °C ± 0.7 °C, respectively. These results suggested that tRNAAla molecule with G37 may be more stable than tRNAAla with A37.
      Figure thumbnail gr3
      Figure 3In vitro analysis for conformation and stability of tRNAAla. A, melting profiles of WT and MT tRNAAla transcripts measured at 260 nm with a heating rate of 1°/min from 25 to 95°. First derivative (dA/dT) against temperature curves was shown to highlight the Tm value transitions. B, northern blot analysis of tRNAs under native conditions. Ten micrograms of total cellular RNA from various cell lines was electrophoresed through native polyacrylamide gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probes specific for the tRNAAla, tRNAThr, tRNAHis, tRNATyr, and 5S rRNA, respectively. C, northern blot analysis of tRNA under denaturing condition. Ten micrograms of total cellular RNAs from the various cell lines was electrophoresed through a 10% denaturing polyacrylamide gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probes specific for tRNAAla, other five tRNAs and 5S rRNA, respectively. D, quantification of tRNA levels. Average relative each tRNA content per cell was normalized to the average content per cell of 5S rRNA in the control and mutant cybrids, respectively. The values for the latter were expressed as percentages of the average values for the control cybrids. The calculations were based on three independent determinations in each cybrids. The error bars indicate two SEM. P indicates the significance, according to the t-test, of the difference between mutant and control cybrids.
      To test if the m.5587A>G mutation affected the conformation of tRNAAla in vivo, total RNAs isolated from mutant and control cell lines were electrophoresed through 10% native gel and then electroblotted onto a positively charged nylon membrane for hybridization analysis with oligodeoxynucleotide probes for tRNAAla, tRNAThr, tRNAHis, and tRNATyr, respectively (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ). As shown in Figure 3B, electrophoretic patterns showed that the tRNAAla in three mutant cybrid cell lines carrying the m.5587A>G mutation migrated faster than those of control cybrid cell lines lacking this mutation. These data indicated the m.5587A>G mutation resulted in the conformation change of tRNAAla.

      Reductions in the steady-state levels of tRNAAla

      To further assess if the m.5587A>G mutation ablated the stability of tRNAAla, we subjected mitochondrial RNAs from mutant and control cybrids to Northern blots through 10% denature gel and hybridized them with DIG-labeled oligodeoxynucleotide probes for 22 tRNAs and 5S rRNA as the loading control (
      • Xiao Y.
      • Wang M.
      • He Q.
      • Xu L.
      • Zhang Q.
      • Meng F.
      • Jia Z.
      • Zhang F.
      • Wang H.
      • Guan M.X.
      Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts.
      ,
      • Zhao X.
      • Cui L.
      • Xiao Y.
      • Mao Q.
      • Aishanjiang M.
      • Kong W.
      • Liu Y.
      • Chen H.
      • Hong F.
      • Jia Z.
      • Wang M.
      • Jiang P.
      • Guan M.X.
      Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript.
      ). As shown in Figure 3C, the steady-state level of tRNAAla in the mutant cells was markedly decreased, as compared with those in control cells. For comparison, the average levels of each tRNA in the various control or mutant cybrids were normalized to the average levels in the same cell lines for reference 5S rRNA. As shown in Figure 3D, the average level of tRNAAla in the mutant cybrid cell lines was 57.83% (p = 0.011) of those in control cell lines, respectively. However, the average levels of other 21 tRNAs in three mutant cybrids were comparable with those in three control cybrids (Fig. 3, C and D, Fig. S2).

      Deficient aminoacylation of tRNAAla

      The A73 is the discriminator base for its cognate aminoacyl-tRNA synthetase (
      • Wende S.
      • Bonin S.
      • Götze O.
      • Betat H.
      • Mörl M.
      The identity of the discriminator base has an impact on CCA addition.
      ). To evaluate if the m.5587A>G mutation affected the aminoacylation of tRNA, we examined the aminoacylation levels of tRNAAla, tRNAThr, tRNAHis, and tRNATyr by the use of electrophoresis in an acidic urea PAGE system to separate uncharged tRNA species from the corresponding charged tRNA, electroblotting and hybridizing with the tRNA probes as described above (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ,
      • Enriquez J.A.
      • Attardi G.
      Analysis of aminoacylation of human mitochondrial tRNAs.
      ). As shown in Figure 4A, the upper band represents the charged tRNA, and the lower band represents uncharged tRNA. The electrophoretic mobility of either charged or uncharged tRNAAla in the mutant cell lines migrated faster than those of control cell lines. To further distinguish nonaminoacylated tRNA from aminoacylated tRNA, samples of tRNAs were deacylated by being heated for 10 min at 60 °C (pH 8.3) and then run in parallel. Only one band (uncharged tRNA) was present in both mutant and control cell lines after deacylating. However, there were no obvious differences in electrophoretic mobility of tRNAThr, tRNAHis, and tRNATyr. As shown in Figure 4B, the efficiencies of aminoacylated tRNAAla in three mutant cell lines were 75.4% (p < 0.001) relative to the average values of three control cell lines. However, the levels of aminoacylation in tRNAThr, tRNAHis, and tRNATyr in mutant cell lines were comparable with those in the control cell lines.
      Figure thumbnail gr4
      Figure 4In vivo aminoacylation assays. A, ten micrograms of total cellular RNA purified from six cell lines under acid conditions was electrophoresed at 4 °C through an acid (pH 5.0) 10% polyacrylamide-8 M urea gel, electroblotted, and hybridized with a DIG-labeled oligonucleotide probe specific for the tRNAAla. The blots were then stripped and rehybridized with tRNAThr, tRNAHis, and tRNATyr, respectively. The samples from one control (C15-T2) and mutant (III2-T1) cell lines were deacylated (DA) by heating for 10 min at 60 °C at pH 8.3 and electrophoresed as above. Aminoacylation assays for tRNAAla were carried out in parallel for aminoacylated and deacylated samples. B, in vivo aminoacylated proportions of tRNAs in the mutant and controls. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to the .

      Decreases in the levels of mitochondrial proteins

      To investigate whether the m.5587A>G mutation impaired mitochondrial translation, western blot analysis was carried out to examine the steady-state levels of 11 mtDNA-encoded polypeptides [ND1, ND3, ND4, ND5, and ND6 (subunits 1, 3, 4, 5, and 6 of NADH dehydrogenase), CYTB (apocytochrome b), CO1, CO2, CO3 (subunits I, II, and III of cytochrome c oxidase), and ATP6, ATP8 (ATPase)] in mutant and control cybrids with GAPDH as a loading control. As shown in Figure 5, A and B, the overall levels of 11 mtDNA-encoded polypeptides in the mutant cell lines were 69.8% (p < 0.001), relative to the mean values measured in the control cell lines. The average levels of ND1, ND3, ND4, ND5, ND6, CO1, CO2, CO3, CYTB, ATP6, and ATP8 in three mutant cybrids were 66.4%, 74%, 55.1%, 65.1%, 73%, 81.5%, 63%, 70%, 52.4%, 66.8%, and 100.9% of those in three control cybrids after normalization to GAPDH, respectively. As shown in Table S2, the reduced levels among 11 mtDNA-encoding polypeptides in the mutant cybrids bearing the 5587A>G mutations were correlated with the proportion of alanine in the polypeptides.
      Figure thumbnail gr5
      Figure 5Analysis of mitochondrial proteins. A, twenty micrograms of total cellular proteins from various cell lines was electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with 11 mtDNA encoding polypeptides in mutant and control cells with GAPDH as a loading control. B, quantification of mitochondrial protein levels. Average relative ND1, ND3, ND4, ND5, ND6, CO1, CO2, CO3, ATP6, ATP8, and CYTB content per cell, normalized to the average content per cell of GAPDH in three mutant cell lines carrying the m.5587A>G mutation and three control cell lines lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell line. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to .

      Deficient activities of respiratory chain complexes

      To evaluate the effect of the m.5587A>G mutation on the oxidative phosphorylation, we measured the activities of respiratory complexes by the use of isolating mitochondria from mutant and control cell lines. The activity of complex I (NADH ubiquinone oxidoreductase) was determined through the oxidation of NADH with ubiquinone as the electron acceptor (
      • Scheffler I.E.
      Mitochondrial disease associated with complex I (NADH-CoQ oxidoreductase) deficiency.
      ). Complex II (succinate ubiquinone oxidoreductase) was examined by the activity of complex II through the artificial electron acceptor DCPIP. The activity of complex III (ubiquinone cytochrome c oxidoreductase) was measured through the reduction of cytochrome c (III) by using D-ubiquinol-2 as the electron donor. The activity of complex IV (cytochrome c oxidase) was monitored through the oxidation of cytochrome c (II). As shown in Figure 6A, the activity of complexes I, III, and IV in three mutant cybrids carrying the m.5587A>G mutation were 71.2% (p = 0.006), 85.1% (p = 0.013), and 77.6% (p = 0.008) of the mean values measured in three control cybrids, respectively, while the activity of complexe II in three mutant cybrids carrying the m.5587A>G mutation was 107.1% (p = 0.388) of the mean values measured in three control cybrids.
      Figure thumbnail gr6
      Figure 6Analysis of enzymatic activities in the OXPHOS complex. A, the enzymatic activities of electron transport chain complexes were investigated by enzymatic assay on complexes I, II, III, and IV in isolated mitochondrial membranes from three mutant and control cybrid cell lines. B, an analysis of O2 consumption in the various cell lines using different inhibitors. The rates of O2 (OCR) were first measured on 2 × 104 cells of each cell line under basal condition and then sequentially added to oligomycin (1.5 μM), FCCP (0.5 μM), rotenone (1 μM), and antimycin A (1 μM) at indicated times to determine different parameters of mitochondrial functions. C, graphs presented the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and nonmitochondrial OCR in mutant and control cell lines. Nonmitochondrial OCR was determined as the OCR after rotenone/antimycin A treatment. Basal OCR was determined as OCR before oligomycin minus OCR after rotenone/antimycin A. ATP-lined OCR was determined as OCR before oligomycin minus OCR after oligomycin. Proton leak was determined as basal OCR minus ATP-linked OCR. Maximal was determined as the OCR after FCCP minus nonmitochondrial OCR. Reserve capacity was defined as the difference between maximal OCR after FCCP minus basal OCR. The average of three determinations for each cell line is shown, the horizontal dashed lines represent the average value for each group. Graph details and symbols are explained in the legend to .
      We then measured oxygen consumption rates (OCR) of various mutant and control cell lines using Seahorse Bioscience XF-96 Extracellular Flux Analyzer (
      • Dranka B.P.
      • Benavides G.A.
      • Diers A.R.
      • Giordano S.
      • Zelickson B.R.
      • Reily C.
      • Zou L.
      • Chatham J.C.
      • Hill B.G.
      • Zhang J.
      • Landar A.
      • Darley-Usmar V.M.
      Assessing bioenergetic function in response to oxidative stress by metabolic profiling.
      ). As shown in Figure 6, B and C, the basal OCR in three mutant cell lines was 73.9% (p = 0.008) relative to the mean values measured in three control cell lines. To investigate which of the enzyme complexes of the respiratory chain was affected in the mutant cell lines, oligomycin (to inhibit the ATP synthase), carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP) (to uncouple the mitochondrial inner membrane and allow for maximum electron flux through the ETC), rotenone (to inhibit complex I), and antimycin A (to inhibit complex III) were added sequentially while measuring OCR. The difference between the basal OCR and the drug-insensitive OCR yields the amount of ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and nonmitochondrial OCR. As shown in Figure 6, B and C, the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity, and nonmitochondrial OCR in three mutant cell lines were 67.5% (p = 0.003), 85.7% (p = 0.328), 74.4% (p = 0.006), 96.7% (p = 0.947), and 88.4% (p = 0.039), relative to the mean values measured in three control cell lines, respectively.

      Instability of OXPHOS complexes

      We analyzed the consequence of m.5587A>G mutation on the stability and activity of complexes I, II, and IV using the in-gel activity assay. Mitochondrial membrane proteins isolated from mutant and control cell lines were separated by blue native electrophoresis analysis (BN-PAGE) and stained with specific substrates of complexes I, II, and IV (
      • Jha P.
      • Wang X.
      • Auwerx J.
      Analysis of mitochondrial respiratory chain super-complexes using blue native polyacrylamide gel electrophoresis (BN-PAGE).
      ,
      • Jin X.
      • Zhang Z.
      • Nie Z.
      • Wang C.
      • Meng F.
      • Yi Q.
      • Chen M.
      • Sun J.
      • Zou J.
      • Jiang P.
      • Guan M.X.
      An animal model for mitochondrial tyrosyl-tRNA synthetase deficiency reveals links between oxidative phosphorylation and retinal function.
      ). As illustrated in Figure 7A, mutant cybrids carrying m.5587A>G mutation exhibited defective assembly of intact supercomplexes and complex I. As shown in Figure 7B, the in-gel activities of complexes I and IV in mutant cell lines carrying m.5587A>G mutation were 72% (p = 0.004) and 82.2% (p < 0.001), relative to the average values of control cell lines, respectively. In contrast, the average in-gel activities of complexes II in the mutant cell lines were comparable with those of the control cell lines.
      Figure thumbnail gr7
      Figure 7BN-PAGE analysis of OXPHOS complexes, measurement of ATP levels and membrane potential. A, in-gel activity of complexes I, II, and IV. Twenty micrograms of mitochondrial proteins from various mutant and control cell lines was used for BN-PAGE, and the activities of complexes were measured in the presence of specific substrates (NADH and NTB for complex I, sodium succinate, phenazine methosulfate, and NTB for complex II, and DAB and cytochrome c for complex IV). B, quantification of in-gel activities of complexes I, II, and IV. The calculations were based on three independent determinations in each cell line. C, measurement of cellular and mitochondrial ATP levels using bioluminescence assay. ATP levels from mutant and control cell lines were measure using a luciferin/luciferase assay. Mutant and control cell lines were incubated with 10 mM glucose or 5 mM 2-deoxy-D-glucose plus 5 mM pyruvate to determine ATP generation under mitochondrial ATP synthesis. Average rates of ATP level per cell line in mitochondria are shown. D, mitochondrial membrane potential analysis. The mitochondrial membrane potential (ΔΨm) was measured in mutant and control cell lines using a fluorescence probe JC-10 assay system. The ratio of fluorescence intensities Ex/Em = 490/590 nm and 490/530 nm (FL590/FL530) was recorded to delineate the ΔΨm level of each sample. The relative ratios of FL590/FL530 geometric mean between m.5587A>G mutation and control cell lines were calculated to reflect the level of ΔΨm. Relative ratio of JC-10 fluorescence intensities at Ex/Em = 490/525 and 490/590 nm in the absence and presence of 10 μM of FCCP in three control cell lines and three mutant cell lines. The average of three determinations for each cell line is shown. Graph details and symbols are explained in the legend to .

      Reduced levels in mitochondrial ATP production

      We used the luciferin/luciferase assay to examine the capacity of oxidative phosphorylation in mutant and wild-type cell lines. Populations of cells were incubated in the media in the presence of glucose and 2-deoxy-D-glucose with pyruvate (
      • Gong S.
      • Peng Y.
      • Jiang P.
      • Wang M.
      • Fan M.
      • Wang X.
      • Zhou H.
      • Li H.
      • Yan Q.
      • Huang T.
      • Guan M.X.
      A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.
      ). As shown in Figure 7C, the levels of ATP production in mutant cell lines in the presence of glucose (total cellular levels of ATP) were comparable with those measured in control cell lines. In contrast, the levels of ATP production in mutant cell lines, in the presence of 2-deoxy-D-glucose and pyruvate to inhibit the glycolysis (mitochondrial levels of ATP), varied from 68% to 75.5%, with an average of 72.1% relative to the mean values measured in the control cell lines (p < 0.001).

      Decrease in mitochondrial membrane potential

      The mitochondrial membrane potentials (ΔΨm) were measured through the fluorescence probe JC-10 assay system in three mutant and three control cell lines (Fig. S3) (
      • Reers M.
      • Smiley S.T.
      • Mottola-Hartshorn C.
      • Chen A.
      • Lin M.
      • Chen L.B.
      Mitochondrial membrane potential monitored by JC-1 dye.
      ). The ratios of fluorescence intensities of Ex/Em = 490/590 and 490/530 nm (FL590/FL530) were recorded to delineate the ΔΨm of each sample. The relative ratios of FL590/FL530 geometric mean between mutant and control cell lines were calculated to represent the level of ΔΨm54. As shown in Figure 7D, the ΔΨm of three mutant cell lines carrying the m.5587A>G mutation ranged from 78.4% to 81.8%, with an average of 79.6% (p < 0.001) of the mean values measured in three control cell lines. In contrast, the levels of ΔΨm in mutant cell lines in the presence of FCCP were comparable with those of control cell lines.

      The increase of ROS production

      Mitochondrial ROS plays a critical role in physiological consequences (
      • Mahfouz R.
      • Sharma R.
      • Lackner J.
      • Aziz N.
      • Agarwal A.
      Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa.
      ,
      • Gong S.
      • Wang X.
      • Meng F.
      • Cui L.
      • Yi Q.
      • Zhao Q.
      • Cang X.
      • Cai Z.
      • Mo J.Q.
      • Liang Y.
      • Guan M.X.
      Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.
      ,
      • Schieber M.
      • Chandel N.S.
      ROS function in redox signaling and oxidative stress.
      ). We assessed ROS production in mutant and control cybrid cell lines via flow cytometry, comparing baseline staining intensity for each cell line with that upon oxidative stress to obtain a ratio corresponding to ROS generation (
      • Jiang P.
      • Wang M.
      • Xue L.
      • Xiao Y.
      • Yu J.
      • Wang H.
      • Yao J.
      • Liu H.
      • Peng Y.
      • Liu H.
      • Li H.
      • Chen Y.
      • Guan M.X.
      A hypertension-associated tRNAAla mutation alters tRNA metabolism and mitochondrial function.
      ,
      • Jin X.
      • Zhang Z.
      • Nie Z.
      • Wang C.
      • Meng F.
      • Yi Q.
      • Chen M.
      • Sun J.
      • Zou J.
      • Jiang P.
      • Guan M.X.
      An animal model for mitochondrial tyrosyl-tRNA synthetase deficiency reveals links between oxidative phosphorylation and retinal function.
      ,
      • Jia Z.
      • Zhang Y.
      • Li Q.
      • Ye Z.
      • Liu Y.
      • Fu C.
      • Cang X.
      • Wang M.
      • Guan M.X.
      A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.
      ). Geometric mean intensity was recorded to measure the rate of mitochondrial ROS of each sample. The relative levels of geometric mean intensity in each cell line were calculated to delineate the levels of mitochondrial ROS in mutant and control cells. As shown in Figure 8, A and B, the levels of ROS generation in the mutant cybrids carrying the m.5587A>G mutation ranged from 118.8% to 127.4%, with an average of 123.4% (p = 0.007) of the mean values measured in the control cell lines. Furthermore, we examined the levels of catalase and superoxide dismutase proteins (SOD2 and SOD1) in mutant and control cell lines by western blot analysis (
      • Gong S.
      • Wang X.
      • Meng F.
      • Cui L.
      • Yi Q.
      • Zhao Q.
      • Cang X.
      • Cai Z.
      • Mo J.Q.
      • Liang Y.
      • Guan M.X.
      Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.
      ,
      • Schieber M.
      • Chandel N.S.
      ROS function in redox signaling and oxidative stress.
      ). As shown in Figure 8, C and D, significant increasing levels of these proteins were observed in the mutant cybrids. In particular, the average levels of catalase, SOD2, and SOD1 in three mutant cell lines carrying the m.5587A>G mutation were 123.7%, 133.4%, and 133%, relative to the mean values measured in three control cell lines, respectively.
      Figure thumbnail gr8
      Figure 8Analysis of mitochondrial ROS production. A and B, the rates of ROS generation by mitochondria in living cells from mutant and control cell lines were analyzed by a Novocyte flow cytometer (ACEA Biosciences) using the mitochondrial superoxide indicator MitoSOX-Red (5 mM). A, flow cytometry histogram showing MitoSOX-Red fluorescence of various cell lines. B, relative ratios of MitoSOX-Red fluorescence intensity. The average of three determinations for each cell line is shown. C, western blot analysis of three antioxidative enzymes. Twenty micrograms of total cellular proteins from various cell lines was electrophoresed, electroblotted, and hybridized with catalase, SOD1, and SOD2 antibodies and with GAPDH as a loading control. D, quantification of SOD2, SOD1, and catalase. Average relative values of SOD2, SOD1, and catalase were normalized to the average values of GAPDH in various cell lines. The values for the latter are expressed as percentages of the average values for the control cell lines. The average of three independent determinations for each cell line is shown. Graph details and symbols are explained in the legend to .

      Promoting apoptosis

      Deficient activities of oxidative phosphorylation have been linked to protection against certain apoptotic stimuli (
      • Jia Z.
      • Zhang Y.
      • Li Q.
      • Ye Z.
      • Liu Y.
      • Fu C.
      • Cang X.
      • Wang M.
      • Guan M.X.
      A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.
      ,
      • Ji Y.
      • Zhang J.
      • Lu Y.
      • Yi Q.
      • Chen M.
      • Xie S.
      • Mao X.
      • Xiao Y.
      • Meng F.
      • Zhang M.
      • Yang R.
      • Guan M.X.
      Complex I mutations synergize to worsen the phenotypic expression of Leber’s hereditary optic neuropathy.
      ). To evaluate if the m.5587A>G mutation affected the apoptotic processes, we examined the apoptotic state of mutant and control cybrids by immunofluorescence and western blot analyses. As shown in Figure 9A, the immunofluorescence patterns of double-labeled cells with rabbit monoclonal antibody specific for the cytochrome c and mouse monoclonal antibody to TOM20 revealed markedly increased levels of cytochrome c in the mutant cells, compared with control cells. The levels of cytochrome c in cytosol in mutant and control cell lines were further evaluated by western blot analysis. As shown in Figure 9, B and C, the levels of cytochrome c in three mutant cell lines ranged from 120.7% to 154.3%, with an average of 141.9% (p = 0.021), relative to the average values in three control cell lines. Furthermore, we examined the levels of four apoptosis activated proteins [caspases 3, 9 and Poly ADP ribose polymerase (PARP)] in mutant and control cell lines by western blot analysis (
      • Ji Y.
      • Zhang J.
      • Lu Y.
      • Yi Q.
      • Chen M.
      • Xie S.
      • Mao X.
      • Xiao Y.
      • Meng F.
      • Zhang M.
      • Yang R.
      • Guan M.X.
      Complex I mutations synergize to worsen the phenotypic expression of Leber’s hereditary optic neuropathy.
      ,
      • Taylor R.C.
      • Cullen S.P.
      • Martin S.J.
      Apoptosis: Controlled demolition at the cellular level.
      ). The average levels of caspases 3, 9 and PARP in three mutant cell lines were 119.3% (p = 0.006), 143% (p = 0.003), and 119.7% (p = 0.018) of the average values measured in three control cell lines, respectively.
      Figure thumbnail gr9
      Figure 9Analysis of apoptosis. A, the distributions of cytochrome c from cybrids (III2-T1 and C15-T2) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and cytochrome c antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were identified by their blue fluorescence. B, western blot analysis of cytochrome c and three apoptosis-activated proteins. Twenty micrograms of total proteins from various cell lines was electrophoresed, electroblotted, and hybridized with cytochrome c, caspases 9 and 3, and PARP antibodies, with GAPDH as a loading control. C, quantification of cytochrome c and three apoptosis-activated proteins. Three independent determinations were done in each cell line. Graph details and symbols are explained in the legend to .

      Alteration in mitophagy

      Mitophagy is the selective removal of damaged mitochondria by autophagosomes and their subsequent catabolism by lysosomes (
      • Anding A.L.
      • Baehrecke E.H.
      Cleaning house: Selective autophagy of organelles.
      ,
      • Korolchuk V.I.
      • Menzies F.M.
      • Rubinsztein D.C.
      A novel link between autophagy and the ubiquitin-proteasome system.
      ,
      • Zhu Y.
      • Chen G.
      • Chen L.
      • Zhang W.
      • Feng D.
      • Liu L.
      • Chen Q.
      Monitoring autophagy in mammalian cells.
      ). To investigate if the m.5587A>G mutation affected the mitophagy, we evaluated the mitophagic states of mutant and control cell lines using endogenous immunofluorescence and western blotting assays. As shown in Figure 10A, mutant cell lines displayed reduced levels of LAMP1 (lysosome-associated membrane glycoprotein 1), indicating that the m.5587A>G impaired the mitophagy process (
      • Anding A.L.
      • Baehrecke E.H.
      Cleaning house: Selective autophagy of organelles.
      ). The status of mitophagy in mutant and control cell lines was then examined using western blot analysis using two markers: microtubule-associated protein 1A/1B light-chain 3B (LC3) and sequestosome 1 (SQSTM1/p62) (
      • Korolchuk V.I.
      • Menzies F.M.
      • Rubinsztein D.C.
      A novel link between autophagy and the ubiquitin-proteasome system.
      ,
      • Zhu Y.
      • Chen G.
      • Chen L.
      • Zhang W.
      • Feng D.
      • Liu L.
      • Chen Q.
      Monitoring autophagy in mammalian cells.
      ). During autophagy, the cytoplasmic form (LC3-I) is processed into a cleaved and lipidated membrane-bound form (LC3-II), which is essential for membrane biogenesis and closure of the membrane. LC3-II is recleaved by cysteine protease (Atg4B) following completion of the autophagosome and recycled. SQSTM1/p62, one of the best-known autophagic substrates, interacts with LC3 to ensure the selective delivery of these proteins into the autophagosome (
      • Zhu Y.
      • Chen G.
      • Chen L.
      • Zhang W.
      • Feng D.
      • Liu L.
      • Chen Q.
      Monitoring autophagy in mammalian cells.
      ). As shown in Figure 10, B and C, the reduced levels of LC3 and increased levels of p62 were observed in the mutant cybrids carrying the m.5587A>G mutation, compared with those in the control cybrids. In particular, the average levels of LC3-II/(LC3-I+II) and p62 in three mutant cell lines carrying the m.5587A>G mutation were 162.5% (p = 0.001) and 66.1% (p = 0.002) of the mean values measured in three control cell lines lacking the mutation, respectively. These data suggested that the m.5587A>G mutation promoted the mitophagy in mutant cybrids.
      Figure thumbnail gr10
      Figure 10Assessment of autophagy. A, the distributions of LAMP1 from mutant (III2-T1) and control (C15-T2) cell lines were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and LAMP1 antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were shown by the blue fluorescence. B, western blot analysis for autophagic response protein LC3-I/II and p62. Twenty micrograms of total cellular proteins from various cell lines was electrophoresed, electroblotted, and hybridized with LC3 and p62 antibodies and with β-actin as a loading control. C, quantification of autophagy markers LC3A/B and p62 in the mutant cell lines and control cell lines. Graph details and symbols are explained in the legend to .

      Discussion

      LHON is the most common type of maternally transmitted eye disorder and is characterized by the degeneration of retinal ganglion cells (RGC) and loss of central vision (
      • Wallace D.C.
      • Lott M.T.
      Leber hereditary optic neuropathy: Exemplar of an mtDNA disease.
      ). In the majority of cases worldwide, LHON was caused by three primary mtDNA point mutations: ND4 11778G>A, ND6 14484T>C, and ND1 3460G>A, affecting subunits of complex I (
      • Wallace D.C.
      • Lott M.T.
      Leber hereditary optic neuropathy: Exemplar of an mtDNA disease.
      ,
      • Wallace D.C.
      • Singh G.
      • Lott M.T.
      • Hodge J.A.
      • Schurr T.G.
      • Lezza A.M.
      • Elsas L.J.
      • Nikoskelainen E.K.
      Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy.
      ,
      • Jiang P.
      • Liang M.
      • Zhang J.
      • Gao Y.
      • He Z.
      • Yu H.
      • Zhao F.
      • Ji Y.
      • Liu X.
      • Zhang M.
      • Fu Q.
      • Tong Y.
      • Sun Y.
      • Zhou X.
      • Huang T.
      • et al.
      Prevalence of mitochondrial ND4 mutations in 1281 Han Chinese subjects with Leber’s hereditary optic neuropathy.
      ,
      • Ji Y.
      • Liang M.
      • Zhang J.
      • Zhu L.
      • Zhang Z.
      • Fu R.
      • Liu X.
      • Zhang M.
      • Fu Q.
      • Zhao F.
      • Tong Y.
      • Sun Y.
      • Jiang O.
      • Guan M.X.
      Mitochondrial ND1 variants in 1281 Chinese subjects with Leber’s hereditary optic neuropathy.
      ,
      • Liang M.
      • Jiang P.
      • Li F.
      • Zhang J.
      • Ji Y.
      • He Y.
      • Xu M.
      • Zhu J.
      • Meng X.
      • Zhao F.
      • Tong Y.
      • Liu X.
      • Sun Y.
      • Zhou X.
      • Mo J.Q.
      • et al.
      Frequency and spectrum of mitochondrial ND6 mutations in 1218 Han Chinese subjects with Leber’s hereditary optic neuropathy.
      ). In the present study, we investigated the pathophysiology of the first LHON-linked tRNA mutation: tRNAAla 5587A>G mutation. This mutation was only present in the matrilineal relatives of three Chinese families with suggestive maternal inheritance of LHON. The occurrence of m.5587A>G mutation in these genetically unrelated pedigrees affected by LHON and differing considerably in their mtDNA sequences strongly indicated that this mutation is involved in the pathogenesis of LHON (
      • Ji Y.
      • Qiao L.
      • Liang X.
      • Zhu L.
      • Gao Y.
      • Zhang J.
      • Jia Z.
      • Wei Q.P.
      • Liu X.
      • Jiang P.
      • Guan M.X.
      Leber’s hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees.
      ). It was anticipated that the m.5587A>G mutation altered the structure and function of tRNAAla. The in silico analysis suggested that the m.5587A>G mutation resulted in an alternate secondary structure fold of the tRNA transcript (
      • Miao Z.
      • Westhof E.
      RNA structure: Advances and assessment of 3D structure prediction.
      ). The altered structure of tRNAAla caused by the m.5587A>G mutation was evidenced by the increased melting temperature of mutated tRNA with respect to the wild-type molecule in vitro transcripts, as in the case of tRNAHis U68>C68 and tRNAAla A1>G1) mutations (
      • Jiang P.
      • Wang M.
      • Xue L.
      • Xiao Y.
      • Yu J.
      • Wang H.
      • Yao J.
      • Liu H.
      • Peng Y.
      • Liu H.
      • Li H.
      • Chen Y.
      • Guan M.X.
      A hypertension-associated tRNAAla mutation alters tRNA metabolism and mitochondrial function.
      ,
      • Gong S.
      • Peng Y.
      • Jiang P.
      • Wang M.
      • Fan M.
      • Wang X.
      • Zhou H.
      • Li H.
      • Yan Q.
      • Huang T.
      • Guan M.X.
      A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.
      ). The potentially altered structure of tRNAAla was responsible for the differential tRNA migration on native gels of mutated tRNA with respect to the wild-type molecule in vitro or ex vivo.
      Here, we demonstrated that the m. 5587A>G mutation had pleiotropic effects on the maturation of tRNAAla. The primary defect arising from the m.5587A>G mutation was the aberrant 3’ end processing of tRNAAla from the L-strand transcripts harboring the ND6 and eight tRNAs including tRNAAla (
      • Xiao Y.
      • Wang M.
      • He Q.
      • Xu L.
      • Zhang Q.
      • Meng F.
      • Jia Z.
      • Zhang F.
      • Wang H.
      • Guan M.X.
      Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts.
      ,
      • Mercer T.R.
      • Neph S.
      • Dinger M.E.
      • Crawford J.
      • Smith M.A.
      • Shearwood A.M.
      • Haugen E.
      • Bracken C.P.
      • Rackham O.
      • Stamatoyannopoulos J.A.
      • Filipovska A.
      • Mattick J.S.
      The human mitochondrial transcriptome.
      ,
      • Ojala D.
      • Montoya J.
      • Attardi G.
      tRNA punctuation model of RNA processing in human mitochondria.
      ). The tRNA processing defects were evidenced by reduced efficiencies of the 3’ end processing of tRNAAla precursors carrying the m.5587A>G mutation using in vitro processing assay and decreased levels of tRNAAla observed in the mutant cells bearing the m.5587A>G mutation. By contrast, the m.5587A>G mutation did not affect the processing of 14 tRNAs, which are cotranscribed from the L-strand mtDNA, and other seven tRNAs, which are cotranscribed from the H-strand mtDNA (
      • Xiao Y.
      • Wang M.
      • He Q.
      • Xu L.
      • Zhang Q.
      • Meng F.
      • Jia Z.
      • Zhang F.
      • Wang H.
      • Guan M.X.
      Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts.
      ,
      • Mercer T.R.
      • Neph S.
      • Dinger M.E.
      • Crawford J.
      • Smith M.A.
      • Shearwood A.M.
      • Haugen E.
      • Bracken C.P.
      • Rackham O.
      • Stamatoyannopoulos J.A.
      • Filipovska A.
      • Mattick J.S.
      The human mitochondrial transcriptome.
      ,
      • Ojala D.
      • Montoya J.
      • Attardi G.
      tRNA punctuation model of RNA processing in human mitochondria.
      ,
      • Zhao X.
      • Cui L.
      • Xiao Y.
      • Mao Q.
      • Aishanjiang M.
      • Kong W.
      • Liu Y.
      • Chen H.
      • Hong F.
      • Jia Z.
      • Wang M.
      • Jiang P.
      • Guan M.X.
      Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript.
      ). Addition of CCA triplet to the 3’ end of mitochondrial tRNAs catalyzed by the enzyme TRNT1 is an important step in the tRNA maturation process and essential for translation (
      • Nagaike T.
      • Suzuki T.
      • Tomari Y.
      • Takemoto-Hori C.
      • Negayama F.
      • Watanabe K.
      • Ueda T.
      Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases.
      ,
      • Shi P.Y.
      • Maizels N.
      • Weiner A.M.
      CCA addition by tRNA nucleotidyltransferase: Polymerization without translocation?.
      ,
      • Hou Y.M.
      CCA addition to tRNA: Implications for tRNA quality control.
      ). Here, we showed that the m.5587A>G mutation significantly inhibited the CCA addition of tRNAAla catalyzed by TRNT1. In fact, the substitution of C72 with U72 in the tRNALeu(UUR) by m.3303C>T mutation led to a significant effect on CCA addition of this tRNA (
      • Levinger L.
      • Giegé R.
      • Florentz C.
      Pathology-related substitutions in human mitochondrial tRNAIle reduce precursor 3’ end processing efficiency in vitro.
      ). Furthermore, the discriminator base A73 at the accepter stem of tRNAAla is critical for its specific recognition by cognate aminoacyl-tRNA synthetase (
      • Kuhle B.
      • Chihade J.
      • Schimmel P.
      Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan mitochondrial tRNAs.
      ,
      • Salinas-Giegé T.
      • Giegé R.
      • Giegé P.
      tRNA biology in mitochondria.
      ,
      • Wende S.
      • Bonin S.
      • Götze O.
      • Betat H.
      • Mörl M.
      The identity of the discriminator base has an impact on CCA addition.
      ,
      • Lovato M.A.
      • Chihade J.W.
      • Schimmel P.
      Translocation within the acceptor helix of a major tRNA identity determinant.
      ,
      • Giege R.
      • Sissler M.
      • Florentz C.
      Universal rules and idiosyncratic features in tRNA identity.
      ,
      • Nagan M.C.
      • Beuning P.
      • Musier-Forsyth K.
      • Cramer C.J.
      Importance of discriminator base stacking interactions: Molecular dynamics analysis of A73 microhelixAla variants.
      ). The in vitro aminoacylation assays showed the complete loss of alanylation of tRNAAla mutants with U73 or C73 and marked decreases in alanylation of tRNAAla mutant with G73 (
      • Zeng Q.Y.
      • Peng G.X.
      • Li G.
      • Zhou J.B.
      • Zheng W.Q.
      • Xue M.Q.
      • Wang E.D.
      • Zhou X.L.
      The G3-U70-independent tRNA recognition by human mitochondrial alanyl-tRNA synthetase.
      ). The impact of purine bases at position 73 on alanylation was further supported by the fact that the cell lines bearing the m.5587A>G mutation exhibited reduced efficiency of tRNAAla aminoacylation in the mutant cell lines and faster electrophoretic mobility of mutated tRNA with respect to the wild-type molecules. Indeed, the faster electrophoretic mobility observed in both native and acid conditions was likely due to a defect of tRNA maturation caused by the m.5587A>G mutation. As a result, deficiencies in the tRNAAla 3’ end metabolism then contributed to the lower levels of tRNAAla in the cybrids carrying the m.5587A>G mutation under native and denatured conditions. However, ∼42% reduction in the steady-state level of tRNAAla in mutant cybrids carrying the m.5587A>G mutation was above the proposed threshold level (70% reduction) to produce a clinical phenotype (
      • Guan M.X.
      • Enriquez J.A.
      • Fischel-Ghodsian N.
      • Puranam R.S.
      • Lin C.P.
      • Maw M.A.
      • Attardi G.
      The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression.
      ,
      • Gong S.
      • Peng Y.
      • Jiang P.
      • Wang M.
      • Fan M.
      • Wang X.
      • Zhou H.
      • Li H.
      • Yan Q.
      • Huang T.
      • Guan M.X.
      A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.
      ,
      • Chomyn A.
      • Enriquez J.A.
      • Micol V.
      • Fernandez-Silva P.
      • Attardi G.
      The mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode syndrome-associated human mitochondrial tRNALeu(UUR) mutation causes aminoacylation deficiency and concomitant reduced association of mRNA with ribosomes.
      ). Therefore, the m.5587A>G mutation may be the primary causative evident but itself insufficient to produce the clinical phenotype.
      The m.5587A>G mutation-induced ramifications of tRNAAla metabolisms impaired the synthesis of 13 mtDNA encoding proteins. In the present study, mutant cell lines carrying the m.5587A>G mutation exhibited the variable reductions (average of 30%) in the levels of 11 mtDNA-encoded polypeptides, ranging from sharp reductions (48%) in the levels of CYTB to no reduction of ATP8. Notably, the reduced levels of these polypeptides in mutant cybrids were significantly correlated with the proportions but not number of alanines (Table S2), consistent with what was previously shown in cells carrying the tRNASer(UCN) 7445A>G mutation (
      • Guan M.X.
      • Enriquez J.A.
      • Fischel-Ghodsian N.
      • Puranam R.S.
      • Lin C.P.
      • Maw M.A.
      • Attardi G.
      The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression.
      ). The impairment of mitochondrial protein synthesis perturbed the stabilities and activities of OXPHOS complexes in the mutant cells bearing the m.5587A>G mutation. In particular, impaired synthesis of complex I subunits (ND1, ND3 ND4, ND5, and ND6) and complex IV subunits (CO1, CO2, and CO3) contributed to the deficiencies in the assembly and activity of complexes I and complex IV, respectively. Furthermore, the m.5587A>G mutation-induced oxidative phosphorylation deficiencies were further supported by significant decreases in the basal OCR, ATP-linked OCR, and maximal OCR as well as significant reductions in the level of mitochondrial ATP in mutant cell lines carrying the m.5587A>G mutation, as revealed by those in the cell lines carrying the tRNAAsp 7551A>G, tRNAMet 4435A>G, and tRNAIle 4295A>G mutations (
      • Peng G.X.
      • Zhang Y.
      • Wang Q.Q.
      • Li Q.R.
      • Xu H.
      • Wang E.D.
      • Zhou X.L.
      The human tRNA taurine modification enzyme GTPBP3 is an active GTPase linked to mitochondrial diseases.
      ,
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ,
      • Wang M.
      • Peng Y.
      • Zheng J.
      • Zheng B.
      • Jin X.
      • Liu H.
      • Wang Y.
      • Tang X.
      • Huang T.
      • Jiang P.
      • Guan M.X.
      A deafness-associated tRNAAsp mutation alters the m1G37 modification, aminoacylation and stability of tRNAAsp and mitochondrial function.
      ). As a result, the defective oxidative phosphorylation diminished mitochondrial membrane potentials and elevated the production of ROS and the subsequent failure of cellular energetic processes (
      • Wallace D.C.
      A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: A dawn for evolutionary medicine.
      ). In particular, the effect of ROS overproduction on cellular functions was evidenced by the increasing expression of antioxidant enzymes SOD1, SOD2 and catalase observed in the cells bearing m.5587A>G mutation (
      • Gong S.
      • Wang X.
      • Meng F.
      • Cui L.
      • Yi Q.
      • Zhao Q.
      • Cang X.
      • Cai Z.
      • Mo J.Q.
      • Liang Y.
      • Guan M.X.
      Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.
      ,
      • Raimundo N.
      • Song L.
      • Shutt T.E.
      • McKay S.E.
      • Cotney J.
      • Guan M.X.
      • Gilliland T.C.
      • Hohuan D.
      • Santos-Sacchi J.
      • Shadel G.S.
      Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.
      ). In particular, RGCs are the biggest ATP demand cells in the retina (
      • Country M.W.
      Retinal metabolism: A comparative look at energetics in the retina.
      ,
      • Wong-Riley M.T.
      Energy metabolism of the visual system.
      ). Therefore, the retinal ganglion cells carrying m.5587A>G mutation may be preferentially involved because they are high ATP demand cells and somehow exquisitely sensitive to subtle imbalance in cellular redox state or increased level of free radicals (
      • Raimundo N.
      • Song L.
      • Shutt T.E.
      • McKay S.E.
      • Cotney J.
      • Guan M.X.
      • Gilliland T.C.
      • Hohuan D.
      • Santos-Sacchi J.
      • Shadel G.S.
      Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.
      ,
      • Wong A.
      • Cavelier L.
      • Collins-Schramm H.E.
      • Seldin M.F.
      • McGrogan M.
      • Savontaus M.L.
      • Cortopassi G.A.
      Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells.
      ,
      • Song L.
      • Yu A.
      • Murray K.
      • Cortopassi G.
      Bipolar cell reduction precedes retinal ganglion neuron loss in a complex 1 knockout mouse model.
      ).
      Mitochondrial dysfunction affected the apoptotic sensitivity and mitophagy of cells carrying the LHON-associated mtDNA mutations (
      • Zhang J.
      • Ji Y.
      • Lu Y.
      • Fu R.
      • Xu M.
      • Liu X.
      • Guan M.X.
      Leber's hereditary optic neuropathy (LHON)-associated ND5 12338T>C mutation altered the assembly and function of complex I, apoptosis and autophagy.
      ,
      • Ji Y.
      • Zhang J.
      • Lu Y.
      • Yi Q.
      • Chen M.
      • Xie S.
      • Mao X.
      • Xiao Y.
      • Meng F.
      • Zhang M.
      • Yang R.
      • Guan M.X.
      Complex I mutations synergize to worsen the phenotypic expression of Leber’s hereditary optic neuropathy.
      ,
      • Melser S.
      • Lavie J.
      • Bénard G.
      Mitochondrial degradation and energy metabolism.
      ). In the present investigation, mutant cybrids bearing the m.5587A>G mutation exhibited more apoptotic susceptibility than control cybrids lacking the mutation. These were evidenced by the elevated releases of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3 and PARP in the cybrids carrying the m.5587A>G mutation, as compared with control cybrids. These data demonstrated that m.5587A>G mutation elevated the apoptosis. Mitophagy regulates mitochondrial energy metabolism by controlling the amount and efficiency of the mitochondrial metabolic machinery (
      • Melser S.
      • Lavie J.
      • Bénard G.
      Mitochondrial degradation and energy metabolism.
      ). In particular, the impairment of OXPHOS and alteration of mitochondrial membrane potential affected the mitophagic removal of damaged mitochondria (
      • Lee J.
      • Giodano S.
      • Zhang J.
      Autophagy, mitochondria and oxidative stress: Cross-talk and redox signaling.
      ). The activation of mitophagic machinery is crucial to the complete degradation of mitochondrial components (
      • Youle R.J.
      • Narendra D.P.
      Mechanisms of autophagy.
      ). In the present investigation, we analyzed the effect of m.5587A>G mutation on mitophagy by western blot and endogenous immunofluorescence experiments. The mutant cybrids bearing the m.5587A>G mutation displayed the increasing levels of autophagy. Furthermore, the mutant cybrids harboring the m.5587A>G mutation revealed the decreased levels of p62, which indicated the preferential reduction of autophagic substrates, and increased levels of LC3-II that implied the increasing generation of autophagosome. These data indicated that the m.5587A>G mutation promoted the autophagic degradation of ubiquitinated proteins.
      However, the incomplete penetrance of LHON and relatively mild biochemical defects indicated that the m.5587A>G mutation is a primary factor underlying the development of LHON but is itself insufficient to produce a clinical phenotype. The other genetic or epigenetic factors may contribute to the development of clinical phenotype in the subjects carrying the m.5587A>G mutation (
      • Zhang J.
      • Ji Y.
      • Lu Y.
      • Fu R.
      • Xu M.
      • Liu X.
      • Guan M.X.
      Leber's hereditary optic neuropathy (LHON)-associated ND5 12338T>C mutation altered the assembly and function of complex I, apoptosis and autophagy.
      ,
      • Ji Y.
      • Zhang J.
      • Lu Y.
      • Yi Q.
      • Chen M.
      • Xie S.
      • Mao X.
      • Xiao Y.
      • Meng F.
      • Zhang M.
      • Yang R.
      • Guan M.X.
      Complex I mutations synergize to worsen the phenotypic expression of Leber’s hereditary optic neuropathy.
      ,
      • Yu J.
      • Liang X.
      • Ji Y.
      • Ai C.
      • Liu J.
      • Zhu L.
      • Nie Z.
      • Jin X.
      • Wang C.
      • Zhang J.
      • Zhao F.
      • Mei S.
      • Zhao X.
      • Zhou X.
      • Zhang M.
      • et al.
      PRICKLE3 linked to ATPase biogenesis manifested Leber’s Hereditary Optic Neuropathy.
      ,
      • Jiang P.
      • Jin X.
      • Peng Y.
      • Wang M.
      • Liu H.
      • Liu X.
      • Zhang Z.
      • Ji Y.
      • Zhang J.
      • Liang M.
      • Zhao F.
      • Sun Y.H.
      • Zhang M.
      • Zhou X.
      • Chen Y.
      • et al.
      The exome sequencing identified the mutation in YARS2 encoding the mitochondrial tyrosyl-tRNA synthetase as a nuclear modifier for the phenotypic manifestation of Leber’s hereditary optic neuropathy-associated mitochondrial DNA mutation.
      ). In particular, the vision-specific phenotypes of this tRNA mutation may be attributed to the tissue specificity of OXPHOS via RNA maturation or involvement of nuclear modifier genes (
      • Jin X.
      • Zhang Z.
      • Nie Z.
      • Wang C.
      • Meng F.
      • Yi Q.
      • Chen M.
      • Sun J.
      • Zou J.
      • Jiang P.
      • Guan M.X.
      An animal model for mitochondrial tyrosyl-tRNA synthetase deficiency reveals links between oxidative phosphorylation and retinal function.
      ,
      • Torres A.G.
      • Reina O.
      • Stephan-Otto Attolini C.
      • Ribas de Pouplana L.
      Differential expression of human tRNA genes drives the abundance of tRNA-derived fragments.
      ,
      • Dittmar K.A.
      • Goodenbour J.M.
      • Pan T.
      Tissue-specific differences in human transfer RNA expression.
      ). However, the m.5587A>G mutation is also associated with Leigh syndrome, which most commonly presents as a progressive dysfunction of the central nervous system (
      • Ma Y.Y.
      • Wu T.F.
      • Liu Y.P.
      • Wang Q.
      • Song J.Q.
      • Li X.Y.
      • Shi X.Y.
      • Zhang W.N.
      • Zhao M.
      • Hu L.Y.
      • Yang Y.L.
      • Zou L.P.
      Genetic and biochemical findings in Chinese children with Leigh syndrome.
      ). Changes in RNA maturation profiles may specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress in the different tissues (
      • Zhang Q.
      • He X.
      • Yao S.
      • Lin T.
      • Zhang L.
      • Chen D.
      • Chen C.
      • Yang Q.
      • Li F.
      • Zhu Y.M.
      • Guan M.X.
      Ablation of Mto1 in zebrafish exhibited hypertrophic cardiomyopathy manifested by mitochondrion RNA maturation deficiency.
      ). Alternatively, the tissue heterogeneity may arise from differential expression of tRNA genes, variable activation of the integrated stress response pathway, metabolic changes, and the ability of certain tissues to respond to impaired mitochondrial translation (
      • Agnew T.
      • Goldsworthy M.
      • Aguilar C.
      • Morgan A.
      • Simon M.
      • Hilton H.
      • Esapa C.
      • Wu Y.
      • Cater H.
      • Bentley L.
      • Scudamore C.
      • Poulton J.
      • Morten K.J.
      • Thompson K.
      • He L.
      • et al.
      A Wars2 mutant mouse model displays OXPHOS deficiencies and activation of tissue-specific stress response pathways.
      ,
      • Dogan S.A.
      • Pujol C.
      • Maiti P.
      • Kukat A.
      • Wang S.
      • Hermans S.
      • Senft K.
      • Wibom R.
      • Rugarli E.I.
      • Trifunovic A.
      Tissue-specific loss of DARS2 activates stress responses independently of respiratory chain deficiency in the heart.
      ).
      In summary, our findings convincingly demonstrate the pathogenic mechanism underlying the LHON-associated tRNAAla 5587A>G mutation. The m.5587A>G mutation led to pleiotropic effects on the 3’ end processing of transcript precursor, CCA addition, aminoacylation, and stability of tRNAAla. The m.5587A>G mutation-induced ramifications of tRNAAla metabolism resulted in the decreased synthesis of mtDNA encoding polypeptides and perturbed the assembly and activity of OXPHOS. As a result, this respiratory deficiency gave rise to the decrease of mitochondrial ATP production, mitochondrial membrane potential, and the increasing production of oxidative reactive species. All those alterations consequently elevated the apoptotic cell death and promoted the mitophagy in cells carrying the m.5587A>G mutation, thereby contributing to visual loss. However, the tissue specificity of this pathogenic mtDNA mutation is likely due to the involvement of nuclear modifier genes or tissue-specific differences in tRNA metabolism. Thus, our findings may provide the new insights into the pathophysiology of LHON manifested by deficiency in mitochondrial tRNAAla 3’ end metabolism.

      Experimental procedures

      Subjects and ophthalmic examinations

      Three LHON Chinese Han pedigrees for this study were ascertained at the Eye Clinic of the Hebei Provincial Eye Hospital (Fig. S1) (
      • Ji Y.
      • Qiao L.
      • Liang X.
      • Zhu L.
      • Gao Y.
      • Zhang J.
      • Jia Z.
      • Wei Q.P.
      • Liu X.
      • Jiang P.
      • Guan M.X.
      Leber’s hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees.
      ). This study was in compliance with the Declaration of Helsinki. Informed consent, blood samples, and clinical evaluations were obtained from all participating family members under protocols approved by the Ethic Committees of Zhejiang University. A comprehensive history and physical examination for these participating subjects were performed at length to identify both personal or family medical histories of visual impairment and other clinical abnormalities. The ophthalmic examinations of probands and other members of these families were conducted as detailed previously (
      • Jiang P.
      • Liang M.
      • Zhang J.
      • Gao Y.
      • He Z.
      • Yu H.
      • Zhao F.
      • Ji Y.
      • Liu X.
      • Zhang M.
      • Fu Q.
      • Tong Y.
      • Sun Y.
      • Zhou X.
      • Huang T.
      • et al.
      Prevalence of mitochondrial ND4 mutations in 1281 Han Chinese subjects with Leber’s hereditary optic neuropathy.
      ,
      • Ji Y.
      • Liang M.
      • Zhang J.
      • Zhu L.
      • Zhang Z.
      • Fu R.
      • Liu X.
      • Zhang M.
      • Fu Q.
      • Zhao F.
      • Tong Y.
      • Sun Y.
      • Jiang O.
      • Guan M.X.
      Mitochondrial ND1 variants in 1281 Chinese subjects with Leber’s hereditary optic neuropathy.
      ,
      • Liang M.
      • Jiang P.
      • Li F.
      • Zhang J.
      • Ji Y.
      • He Y.
      • Xu M.
      • Zhu J.
      • Meng X.
      • Zhao F.
      • Tong Y.
      • Liu X.
      • Sun Y.
      • Zhou X.
      • Mo J.Q.
      • et al.
      Frequency and spectrum of mitochondrial ND6 mutations in 1218 Han Chinese subjects with Leber’s hereditary optic neuropathy.
      ). The degree of visual impairment was defined according to the visual acuity as follows: normal > 0.3, mild = 0.3 to 0.1; moderate < 0.1 to 0.05; severe < 0.05 to 0.02; and profound < 0.02 (
      • Qu J.
      • Li R.
      • Tong Y.
      • Lu F.
      • Qian Y.
      • Hu Y.
      • Mo J.Q.
      • West C.E.
      • Guan M.X.
      The novel A4435G mutation in the mitochondrial tRNAMet may modulate the phenotypic expression of the LHON-associated ND4 G11778A mutation.
      ). A total of 485 control DNA samples were obtained from adult Han Chinese from the same area.

      Cell lines and culture conditions

      Immortalized lymphoblastoid cell lines were generated from one affected subject (III2) of the Chinese family (HZL003) carrying the m.5587A>G mutation and one genetically unrelated Chinese control individual (C15) belonging to the same mtDNA haplogroup F1 but lacking the mutation (Supplemental Methods and Table S3) (
      • Miller G.
      • Lipman M.
      Release of infectious Epstein–Barr virus by transformed marmoset leukocytes.
      ). These cell lines were grown in RPMI 1640 medium with 10% fetal bovine serum (FBS). The bromodeoxyuridine (BrdU)-resistant 143B.TK cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo fisher) (containing 4.5 mg of glucose and 0.11 mg pyruvate/ml), supplemented with 100 μg of BrdU/ml and 5% FBS. The mtDNA-less ρo206 cell line, derived from 143B.TKcells, was grown under the same conditions as the parental line, except for the addition of 50 μg of uridine/ml. Transformation by cytoplasts of mtDNA-less ρo206 cells using one affected subject (III2) carrying the m.5587A>G mutation and one control individual was performed as described elsewhere (
      • King M.P.
      • Attadi G.
      Mitochondria-mediated transformation of human ρo cells.
      ,
      • Zhang J.
      • Ji Y.
      • Lu Y.
      • Fu R.
      • Xu M.
      • Liu X.
      • Guan M.X.
      Leber's hereditary optic neuropathy (LHON)-associated ND5 12338T>C mutation altered the assembly and function of complex I, apoptosis and autophagy.
      ). All cybrid cell lines constructed with enucleated lymphoblastoid cell lines were maintained in the same medium as the 143B.TK cell line. An analysis for the presence and level of m.5587A>G mutation was carried out as described previously (Supplemental Methods) (
      • Ji Y.
      • Qiao L.
      • Liang X.
      • Zhu L.
      • Gao Y.
      • Zhang J.
      • Jia Z.
      • Wei Q.P.
      • Liu X.
      • Jiang P.
      • Guan M.X.
      Leber’s hereditary optic neuropathy is potentially associated with a novel m.5587T>C mutation in two pedigrees.
      ). The quantification of mtDNA copy number from different cybrids was performed as detailed previously (
      • Zhang J.
      • Ji Y.
      • Lu Y.
      • Fu R.
      • Xu M.
      • Liu X.
      • Guan M.X.
      Leber's hereditary optic neuropathy (LHON)-associated ND5 12338T>C mutation altered the assembly and function of complex I, apoptosis and autophagy.
      ). Three mutant cybrids (III2-T1, III2-T6, and III2-T8) carrying the m.5587A>G mutation and three control cybrids (C15-T2, C15-T3, and C15-T5) lacking the mutation with similar mtDNA copy numbers and same karyotype were used for the biochemical characterization described below.

      Mitochondrial RNase Z cleavage assay

      The wild-type and mutant precursors of tRNAAla corresponding to mtDNA at positions 5655 (5′) to 5567 (3′) were cloned into the pCRII-TOPO vector carrying SP6 and T7 promoters (Clontech). After HindIII digestion, the labeled RNA substrates (89 nt for tRNAAla) were transcribed with T7 RNA polymerase in the presence of 10 μM ATP, CTP, GTP, and UTP, pH 7.5, and 10 units RNase inhibitor at 20 °C. Transcripts were purified by denaturing 10% polyacrylamide gel electrophoresis (PAGE) [8 M urea, 8% polyacrylamide/bisacrylamide (19:1)] and were dissolved in 1 mM EDTA. Mitochondrial RNase Z was reconstituted from purified recombinant proteins ELAC2 as detailed elsewhere (
      • Reinhard L.
      • Sridhara S.
      • Hallberg B.M.
      The MRPP1/MRPP2 complex is a tRNA-maturation platform in human mitochondria.
      ). The reaction mixtures were incubated in 20 μl assay buffer containing 20 mM HEPES (pH 7.6), 20 mM KCl, 2 mM MgCl2, 2 mM DTT, 0.1 mg/ml bovine serum albumin (BSA), 80uM S-adenosyl-methionine (SAM), 1 U RiboLock RNase Inhibitor (Thermo Fisher Scientific), 300 ng pre-tRNAs, 800 nM ELAC2. After 5, 10, 15, 20, 25, and 40 min, aliquots were withdrawn and stopped by addition of loading buffer (85% formamide, 10 mM EDTA). The reaction products were separated by denaturing 10% PAGE in 1 × Tris-borate-EDTA (TBE) buffer. After electrophoresis, the reaction products were visualized by staining with NA-Red (Beyotime).

      Mitochondrial tRNA CCA-adding activity assays

      The CCA tRNA nucleotidyltransferase assays were carried out in assay buffer containing 20 mM K-HEPES pH 7.6, 30 mM KCl, 5 mM MgCl2, 0.5 mM NTPs, 2 mM DTT, and 0.1 mg/ml BSA using 200 nM pre-tRNAAla samples and 50 nM CCA-adding enzyme. The reaction mixes were preincubated at 30 °C for 10 min, and the reaction was initiated by the addition of CCA-adding enzyme TRNT1 (
      • Jiang P.
      • Wang M.
      • Xue L.
      • Xiao Y.
      • Yu J.
      • Wang H.
      • Yao J.
      • Liu H.
      • Peng Y.
      • Liu H.
      • Li H.
      • Chen Y.
      • Guan M.X.
      A hypertension-associated tRNAAla mutation alters tRNA metabolism and mitochondrial function.
      ). After 5, 10, 15, 20, 25, and 40 min at 30 °C, aliquots were withdrawn and stopped by addition of loading buffer (85% formamide, 10 mM EDTA). The reaction products were separated by denaturing 10% PAGE in 1 × TBE buffer and visualized by staining with NA-Red (Beyotime) (
      • Reinhard L.
      • Sridhara S.
      • Hallberg B.M.
      The MRPP1/MRPP2 complex is a tRNA-maturation platform in human mitochondria.
      ).

      UV melting assays

      UV melting assays were carried out, as described previously (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ,
      • Wang M.
      • Liu H.
      • Zheng J.
      • Chen B.
      • Zhou M.
      • Fan W.
      • Wang H.
      • Liang X.
      • Zhou X.
      • Eriani G.
      • Jiang P.
      • Guan M.X.
      A deafness- and diabetes-associated trna mutation causes deficient pseudouridinylation at position 55 in tRNAGlu and mitochondrial dysfunction.
      ). The wild-type and mutant tRNAAla transcripts were generated as described above. The tRNAAla transcripts were diluted in a buffer including 50 mM sodium phosphate (pH 7.0), 50 mM NaCl, 5 mM MgCl2, and 0.1 mM EDTA. Absorbance against temperature melting curves was measured at 260 nm with a heating rate of 1 °C/min from 25 to 95 °C through Agilent Cary 100 UV Spectrophotometer.

      Mitochondrial tRNA analysis

      RNAs were obtained by using TOTALLY RNA kit (Ambion) from intact cells or mitochondria isolated from mutant and control cell lines (∼2 × 108 cells), as detailed elsewhere (
      • King M.P.
      • Attardi G.
      Post-transcriptional regulation of the steady-state levels of mitochondrial tRNAs in HeLa cells.
      ). For tRNA Northern blot analysis, 10 μg of RNAs was electrophoresed through a 10% polyacrylamide/8 M urea gel in TBE (after heating the sample at 65 °C for 10 min). The gels were then electroblotted onto a positively charged nylon membrane (Roche) for the hybridization analysis with DIG-labeled oligodeoxynucleotide probes for 22 mitochondrial tRNAs and 5S rRNA as detailed previously (
      • Xiao Y.
      • Wang M.
      • He Q.
      • Xu L.
      • Zhang Q.
      • Meng F.
      • Jia Z.
      • Zhang F.
      • Wang H.
      • Guan M.X.
      Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts.
      ,
      • Zhao X.
      • Cui L.
      • Xiao Y.
      • Mao Q.
      • Aishanjiang M.
      • Kong W.
      • Liu Y.
      • Chen H.
      • Hong F.
      • Jia Z.
      • Wang M.
      • Jiang P.
      • Guan M.X.
      Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript.
      ,
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ). DIG-labeled oligodeoxynucleotides were generated by using DIG oligonucleotide Tailing kit (Roche). The hybridization and quantification of density in each band were performed as detailed elsewhere (
      • Xiao Y.
      • Wang M.
      • He Q.
      • Xu L.
      • Zhang Q.
      • Meng F.
      • Jia Z.
      • Zhang F.
      • Wang H.
      • Guan M.X.
      Asymmetrical effects of deafness-associated mitochondrial DNA 7516delA mutation on the processing of RNAs in the H-strand and L-strand polycistronic transcripts.
      ,
      • Zhao X.
      • Cui L.
      • Xiao Y.
      • Mao Q.
      • Aishanjiang M.
      • Kong W.
      • Liu Y.
      • Chen H.
      • Hong F.
      • Jia Z.
      • Wang M.
      • Jiang P.
      • Guan M.X.
      Hypertension-associated mitochondrial DNA 4401A>G mutation caused the aberrant processing of tRNAMet, all 8 tRNAs and ND6 mRNA in the light-strand transcript.
      ,
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ,
      • Jia Z.
      • Zhang Y.
      • Li Q.
      • Ye Z.
      • Liu Y.
      • Fu C.
      • Cang X.
      • Wang M.
      • Guan M.X.
      A coronary artery disease-associated tRNAThr mutation altered mitochondrial function, apoptosis and angiogenesis.
      ).
      For the aminoacylation assays, total cellular RNAs were isolated under acid conditions, and 10 μg of total cellular RNAs was electrophoresed at 4 °C through an acid (pH 5.0) 10% polyacrylamide/8 M urea gel to separate the charged and uncharged tRNA as detailed elsewhere (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ,
      • Enriquez J.A.
      • Attardi G.
      Analysis of aminoacylation of human mitochondrial tRNAs.
      ). To further distinguish nonaminoacylated tRNA from aminoacylated tRNA, samples of tRNAs were deacylated by being heated for 10 min at 60 °C (pH 8.3) and then run in parallel (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ,
      • Enriquez J.A.
      • Attardi G.
      Analysis of aminoacylation of human mitochondrial tRNAs.
      ). The gels were then electroblotted onto a positively charged nylon membrane (Roche) for the hybridization analysis with oligodeoxynucleotide probes as described above. Quantification of density in each band was performed as detailed previously (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ,
      • Enriquez J.A.
      • Attardi G.
      Analysis of aminoacylation of human mitochondrial tRNAs.
      ).
      For the tRNA mobility shift assay, 10 μg of total cellular RNAs was electrophoresed through a 10% polyacrylamide native gel at 4 °C with 50 mM Tris-glycine buffer. After electrophoresis, the gels were treated according to the Northern blot analysis as described above (
      • Zhou M.
      • Xue L.
      • Chen Y.
      • Li H.
      • He Q.
      • Wang B.
      • Meng F.
      • Wang M.
      • Guan M.X.
      A hypertension-associated mitochondrial DNA mutation introduces an m1G37 modification into tRNAMet, altering its structure and function.
      ).

      Western blot analysis

      Western blotting analysis was performed as detailed elsewhere (
      • Gong S.
      • Peng Y.
      • Jiang P.
      • Wang M.
      • Fan M.
      • Wang X.
      • Zhou H.
      • Li H.
      • Yan Q.
      • Huang T.
      • Guan M.X.
      A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.
      ). Five micrograms of total proteins obtained from lysed mitochondria was denatured and loaded on sodium dodecyl sulfate (SDS) polyacrylamide gels. The gels were electroblotted onto a polyvinylidene difluoride (PVDF) membrane for hybridization. The antibodies used for this investigation were from Abcam [ND5 (ab92624), CO2 (ab110258), catalase (ab52477), cytochrome c (ab133504), P62 (ab56416), p62 (ab56416)], Sigma [ND6 (SAB2108622)], Proteintech Group [ND1 (19703-1-AP), SOD1 (10269-1-AP), SOD2 (24127-1-AP), GAPDH (60004-1-Ig), CYTB (55090-1-AP) and ATP8 (26723-1-AP)], ABclonal Technology [ND3(A9940), CO1 (A17889), CO3 (A9939) and ATP6 (A8193)], Cell Signaling Technology[PARP (9542), caspase 3 (14420), caspase 9 (9508), and LC3I/II (12741)], Novus [ND4 (NBP2-47365)]. Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Beyotime) were used as a secondary antibody and protein signals were detected using the ECL system (Millipore). Quantification of density in each band was performed as detailed previously (
      • Gong S.
      • Peng Y.
      • Jiang P.
      • Wang M.
      • Fan M.
      • Wang X.
      • Zhou H.
      • Li H.
      • Yan Q.
      • Huang T.
      • Guan M.X.
      A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.
      ,
      • Gong S.
      • Wang X.
      • Meng F.
      • Cui L.
      • Yi Q.
      • Zhao Q.
      • Cang X.
      • Cai Z.
      • Mo J.Q.
      • Liang Y.
      • Guan M.X.
      Overexpression of mitochondrial histidyl-tRNA synthetase restores mitochondrial dysfunction caused by a deafness-associated tRNAHis mutation.
      ).

      Blue native electrophoresis analysis

      BN-PAGE was performed by isolating mitochondrial proteins from mutant and control cell lines, as detailed elsewhere (
      • Jha P.
      • Wang X.
      • Auwerx J.
      Analysis of mitochondrial respiratory chain super-complexes using blue native polyacrylamide gel electrophoresis (BN-PAGE).
      ,
      • Ji Y.
      • Zhang J.
      • Yu J.
      • Wang Y.
      • Lu Y.
      • Liang M.
      • Li Q.
      • Jin X.
      • Wei Y.
      • Meng F.
      • Gao Y.
      • Cang X.
      • Tong Y.
      • Liu X.
      • Zhang M.
      • et al.
      Contribution of mitochondrial ND1 3394T>C mutation to the phenotypic manifestation of Leber’s hereditary optic neuropathy.
      ). Samples containing 30 μg of total cellular proteins were separated on 3 to 12% Bis-Tris Native PAGE gel. The native gels were prewashed in cold water and then incubated with the substrates of complex I, complex II, and complex IV at room temperature as described elsewhere (
      • Jin X.
      • Zhang Z.
      • Nie Z.
      • Wang C.
      • Meng F.
      • Yi Q.
      • Chen M.
      • Sun J.
      • Zou J.
      • Jiang P.
      • Guan M.X.
      An animal model for mitochondrial tyrosyl-tRNA synthetase deficiency reveals links between oxidative phosphorylation and retinal function.
      ,
      • Ji Y.
      • Zhang J.
      • Lu Y.
      • Yi Q.
      • Chen M.
      • Xie S.
      • Mao X.
      • Xiao Y.
      • Meng F.
      • Zhang M.
      • Yang R.
      • Guan M.X.
      Complex I mutations synergize to worsen the phenotypic expression of Leber’s hereditary optic neuropathy.
      ). After stopping reaction with 10% acetic acid, gels were washed with water and scanned to visualize the activities of respiratory chain complexes.

      Assays of activities of respiratory chain complexes

      The enzymatic activities of complexes I, II, III, and IV were assayed as detailed elsewhere (
      • Birch-Machin M.A.
      • Turnbull D.M.
      Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues.
      ,
      • Meng F.
      • He Z.
      • Tang X.
      • Zheng J.
      • Jin X.
      • Zhu Y.
      • Ren X.
      • Zhou M.
      • Wang M.
      • Gong S.
      • Mo J.Q.
      • Shu Q.
      • Guan M.X.
      Contribution of the tRNAIle 4317A>G mutation to the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA 1555A>G mutation.
      ).

      Measurements of oxygen consumption

      The OCR in various cybrid cell lines were measured with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience), as detailed previously (
      • Dranka B.P.
      • Benavides G.A.
      • Diers A.R.
      • Giordano S.
      • Zelickson B.R.
      • Reily C.
      • Zou L.
      • Chatham J.C.
      • Hill B.G.
      • Zhang J.
      • Landar A.
      • Darley-Usmar V.M.
      Assessing bioenergetic function in response to oxidative stress by metabolic profiling.
      ,
      • Gong S.
      • Peng Y.
      • Jiang P.
      • Wang M.
      • Fan M.
      • Wang X.
      • Zhou H.
      • Li H.
      • Yan Q.
      • Huang T.
      • Guan M.X.
      A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.
      ). Cells were seeded at a density of 2 × 104 cells per well on Seahorse XF96 polystyrene tissue culture plates (Seahorse Bioscience). Inhibitors were used at the following concentrations: oligomycin (1.5 μM), FCCP (0.8 μM), antimycin A (1.5 μM), and rotenone (3 μM), respectively.

      ATP measurements

      The Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) was used for the measurement of cellular and mitochondrial ATP levels, according to the modified manufacturer's instructions, as described previously (
      • Gong S.
      • Peng Y.
      • Jiang P.
      • Wang M.
      • Fan M.
      • Wang X.
      • Zhou H.
      • Li H.
      • Yan Q.
      • Huang T.
      • Guan M.X.
      A deafness-associated tRNAHis mutation alters the mitochondrial function, ROS production and membrane potential.
      ).

      Assessment of mitochondrial membrane potential

      Mitochondrial membrane potential was assessed with JC-10 Assay Kit-Microplate (Abcam) following general manufacturer’s recommendations with some modifications, as detailed elsewhere (
      • Reers M.
      • Smiley S.T.
      • Mottola-Hartshorn C.
      • Chen A.
      • Lin M.
      • Chen L.B.
      Mitochondrial membrane potential monitored by JC-1 dye.
      ).

      Measurement of ROS production

      ROS measurements were performed following the procedures, as detailed previously (
      • Jiang P.
      • Wang M.
      • Xue L.
      • Xiao Y.
      • Yu J.
      • Wang H.
      • Yao J.
      • Liu H.
      • Peng Y.
      • Liu H.
      • Li H.
      • Chen Y.
      • Guan M.X.
      A hypertension-associated tRNAAla mutation alters tRNA metabolism and mitochondrial function.
      ,
      • Mahfouz R.
      • Sharma R.
      • Lackner J.
      • Aziz N.
      • Agarwal A.
      Evaluation of chemiluminescence and flow cytometry as tools in assessing production of hydrogen peroxide and superoxide anion in human spermatozoa.
      ).

      Immunofluorescence analysis

      Immunofluorescence experiments were undertaken as described elsewhere (
      • Ji Y.
      • Zhang J.
      • Lu Y.
      • Yi Q.
      • Chen M.
      • Xie S.
      • Mao X.
      • Xiao Y.
      • Meng F.
      • Zhang M.
      • Yang R.
      • Guan M.X.
      Complex I mutations synergize to worsen the phenotypic expression of Leber’s hereditary optic neuropathy.
      ). Cells were cultured on cover glass slips (Thermo Fisher), fixed in 4% formaldehyde for 15 min, permeabilized with 0.2% Triton X-100, blocked with 5% FBS for 1 h, and immunostained with TOM20, cytochrome C, and LAMP1 antibodies overnight at 4 °C, respectively. The cells were then incubated with Alex Fluor 594 goat anti-rabbit IgG (H+L) and Alex Fluor 488 goat anti-mouse IgG (H+L) (Thermo Fisher), stained with 4’, 6-diamidino-2-phenylindole (DAPI; Invitrogen) for 15 min and mounted with Fluoromount (Sigma-Aldrich). Cells were examined using a confocal fluorescence microscope (Olympus Fluoview FV1000) with three lasers (Ex/Em = 550/570, 492/520, and 358/461 nm).

      Statistical analysis

      Statistical analysis was carried out using the unpaired, two-tailed Student’s t-test contained in the Microsoft-Excel program or Macintosh (version 2019). Differences were considered significant at a p < 0.05.

      Data availability

      Representative experiments are shown in the Figures and supplemental materials. For any additional information, please contact the corresponding author.

      Supporting information

      This article contains supporting information.

      Conflict of interest

      All authors declare that they have no conflict of interest with contents of this article.

      Acknowledgments

      This work was supported by grants from the National Natural Science Foundation of China (31970557, 82071007); the National Key research and Development Program (2018YFC1004802), Zhejiang Provincial Public Welfare Technology Applied Research Projects (LGF21C060001) and Zhejiang Provincial Traditional Chinese Medicine Research Projects (2019ZB072), Zhejiang Provincial Natural Science Foundation of China (LY19H130005 and LY20C060003), Zhejiang Provincial Health Technology Project Commission (2019RC200), and Wenzhou Science and Technology Bureau (Y20190067). We thank Ms Jiji Sun’s technical assistance.

      Author contributions

      Y. J., Z. N., F. M., C. H., H. C., L. J., M. C., M. Z., J. Z., M. L., and M.-X. G. data curation; Y. J. and M.-X. G. formal analysis; Y. J., Z. N., J. Z., and M. W. validation; Y. J., Z. N., F. M., and L. J. investigation; Y. J. visualization; Y. J., Z. N., F. M., and C. H., methodology; Y. J. and M.-X. G. writing–original draft; M. Z., J. Z., and M.-X. G. resources; M. W. and M.-X. G. project administration; M.-X. G. conceptualization; M.-X. G. supervision; M.-X. G. funding acquisition; M.-X. G. Writing-review and editing.

      Supporting information

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