Human mitochondrial leucyl-tRNA synthetase corrects mitochondrial dysfunctions due to the tRNALeu(UUR) A3243G mutation, associated with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms and diabetes

Abstract

Mutations in mitochondrial tRNA genes are associated with a wide spectrum of human diseases. In particular, the tRNA(Leu(UUR)) A3243G mutation causes mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms (MELAS) and 2% of cases of type 2 diabetes. The primary defect in this mutation was an inefficient aminoacylation of the tRNA(Leu(UUR)). In the present study, we have investigated the molecular mechanism of the A3243G mutation and whether the overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) in the cytoplasmic hybrid (cybrid) cells carrying the A3243G mutation corrects the mitochondrial dysfunctions. Human LARS2 localizes exclusively to mitochondria, and LARS2 is expressed ubiquitously but most abundantly in tissues with high metabolic rates. We showed that the alteration of aminoacylation tRNA(Leu(UUR)) caused by the A3243G mutation led to mitochondrial translational defects and thereby reduced the aminoacylated efficiencies of tRNA(Leu(UUR)) as well as tRNA(Ala) and tRNA(Met). We demonstrated that the transfer of human mitochondrial leucyl-tRNA synthetase into the cybrid cells carrying the A3243G mutation improved the efficiency of aminoacylation and stability of mitochondrial tRNAs and then increased the rates of mitochondrial translation and respiration, consequently correcting the mitochondrial dysfunction. These findings provide new insights into the molecular mechanism of maternally inherited diseases and a step toward therapeutic interventions for these disorders.

FIG. 1.

Subcellular localization and gene expression analysis of human LARS2. (A) Multiple-tissue Northern blot analysis of LARS2 expression. A human 12-lane multiple-tissue blot (Clontech) containing 2 μg of poly(A)⁺ RNA per lane was hybridized with a ³²P-labeled fragment containing a human 1,124-bp LARS2 cDNA probe according to the manufacturer's protocol. The blot was then stripped and rehybridized with ³²P-labeled human β-actin probe as a control. (B) Subcellular localization of human LARS2 in 143B cells. Cells were transiently transfected with a LARS2 cDNA fused with GFP or pEGFP. The fusion protein was visualized by indirect immunofluorescence using antibodies to mouse COX1 and to GFP. A merged image is shown on the right.

FIG. 2.

(A) Construction of stable transfectants. Expression analysis of human LARS2 in the transfectants. Equal amounts (5 μg) of total mitochondrial RNA from mutant cell lines and control cell lines were electrophoresed through a 1.8% agarose-formaldehyde gel, transferred onto a positively charged membrane, and hybridized first with a DIG-labeled LARS2-specific RNA probe. After the blot was stripped, it was hybridized with a human β-actin probe as a control. 43B is a cybrid cell line carrying a nearly homoplasmic A3243G mutation, the HSI cell line is an isogenic wild-type cybrid line derived from the same individuals as those for 43B-V and HSI-V are transfectant lines with vector only, and 43B-LARS2 and HSI-LARS2 are transfectant lines expressing the human LARS2. The 4.2-kb band and 2.8-kb bands correspond to the length of endogenous and exogenous LARS2 mRNA, respectively. (B) Quantification of the A3243G mutation in the tRNA^Leu(UUR) gene in transfectants and parental cybrids. PCR products around the A3243G mutation were digested with ApaI and analyzed by electrophoresis in a 6% polyacrylamide gel stained with ethidium bromide. The A3243G mutation creates the site for restriction enzyme ApaI (14). Transfectants and their parental cybrid cell lines are indicated.

FIG. 3.

In vivo aminoacylation assays. (A) Equal amounts (2 μg) of total mitochondrial RNA purified from six cell lines (the same as in Fig. 2A) under acid conditions were electrophoresed at 4°C through an acid (pH 5.2) 10% polyacrylamide-7 M urea gel, electroblotted, and hybridized with a DIG-labeled oligonucleotide probe specific for the mitochondrial tRNA^Leu(UUR). The blots were then stripped and rehybridized with DIG-labeled tRNA^Leu(UUR), tRNA^Leu(CUN), tRNA^Lys, tRNA^Met, tRNA^Ser(UCN), and tRNA^Ala, respectively. Samples were deacylated (DA) by being heated for 10 min at 60°C at pH 8.3. 43B is a cybrid cell line carrying a nearly homplasmic A3243G mutation, the HSI cell line is an isogenic wild-type cybrid line derived from the same individuals as those for 43B-V and HSI-V are transfectant lines with vector only, and 43B-LARS2 and HSI-LARS2 are transfectant lines expressing the human LARS2. (B) In vivo aminoacylated proportions of tRNA^Leu(UUR), tRNA^Leu(CUN), tRNA^Lys, tRNA^Met, tRNA^Ser(UCN), and tRNA^Ala in the mutant and controls. The calculations were based on three independent determinations. The error bars indicate two standard errors of the means.

FIG. 4.

Assays for the steady-state levels of mitochondrial tRNA^Leu(CUN). (A) Northern blot analysis of mitochondrial tRNA. Equal amounts (2 μg) of total mitochondrial RNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted, and hybridized with DIG-labeled oligonucleotide probes specific for the tRNA^Leu(UUR), tRNA^Leu(CUN), tRNA^Lys, tRNA^Met, tRNA^Ser(UCN), and tRNA^Ala as well as 5S RNA, respectively. (B) Quantification of the mitochondrial tRNA levels. Average relative tRNA^Leu(UUR), tRNA^Leu(CUN), tRNA^Lys, tRNA^Met, tRNA^Ser(UCN), and tRNA^Ala contents per cell were normalized to the average content per cell of 5S RNA in control cell lines and in mutant cell lines, respectively. The values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations of tRNA^Leu(UUR) content in each cell line and three determinations of the content of each reference RNA marker in each cell line. The error bars indicate two standard errors of the means.

FIG. 5.

Expression analysis of mitochondrial RNA. Equal amounts (5 μg) of total mitochondrial RNA from mutant cell lines and control cell lines were electrophoresed through a 1.8% agarose-formaldehyde gel, transferred onto a positively charged membrane, and hybridized first with a DIG-labeled ND1-specific RNA probe. After the blot was stripped, it was hybridized with DIG-labeled RNA probes ND4, ND6, 12S rRNA, and 16S rRNA, respectively. RNA19 consists of 12S rRNA plus tRNA^Val plus 16S rRNA plus tRNA^Leu(UUR) plus ND1 (22, 24).

FIG. 6.

Analysis of rates of mitochondrial protein labeling. (A) Electrophoretic patterns of the mitochondrial translation products of different cell lines labeled for 30 min with [³⁵S]methionine in the presence of 100 μg of emetine per ml. Samples containing equal amounts of protein (20 μg) were run in SDS-polyacrylamide gradient gels. COI, COII, and COIII, subunits I, II, and III of cytochrome c oxidase, respectively; ND1, ND2, ND3, ND4, ND4L, ND5, and ND6, subunits 1, 2, 3, 4, 4L, 5, and 6 of the respiratory-chain NADH dehydrogenase, respectively; A6 and A8, subunits 6 and 8 of the H⁺-ATPase, respectively; CYTB, apocytochrome b. (B) Quantification of the rates of labeling of the mitochondrial translation products. The rates of mitochondrial protein labeling in three mutant cell lines and three control cell lines, determined as described elsewhere (16, 17), are expressed as percentages of the value for HSI in each gel, with error bars indicating standard errors of the means. Three independent labeling experiments and three electrophoretic analyses of each labeled preparation were carried out on each cell line.

FIG. 7.

Respiration assay. Average rates of total O₂ consumption per cell were measured in different cell lines. Four to eight determinations were carried out for each cell line, with error bars indicating standard errors of the means.