An N-terminal formyl methionine on COX 1 is required for the assembly of cytochrome c oxidase.

Protein synthesis in mitochondria is initiated by formylmethionyl-tRNA(Met) (fMet-tRNA(Met)), which requires the activity of the enzyme MTFMT to formylate the methionyl group. We investigated the molecular consequences of mutations in MTFMT in patients with Leigh syndrome or cardiomyopathy. All patients studied were compound heterozygotes. Levels of MTFMT in patient fibroblasts were almost undetectable by immunoblot analysis, and BN-PAGE analysis showed a combined oxidative phosphorylation (OXPHOS) assembly defect involving complexes I, IV and V. The synthesis of only a subset of mitochondrial polypeptides (ND5, ND4, ND1, COXII) was decreased, whereas all others were translated at normal or even increased rates. Expression of the wild-type cDNA rescued the biochemical phenotype when MTFMT was expressed near control levels, but overexpression produced a dominant-negative phenotype, completely abrogating assembly of the OXPHOS complexes, suggesting that MTFMT activity must be tightly regulated. fMet-tRNA(Met) was almost undetectable in control cells and absent in patient cells by high-resolution northern blot analysis, but accumulated in cells overexpressing MTFMT. Newly synthesized COXI was under-represented in complex IV immunoprecipitates from patient fibroblasts, and two-dimensional BN-PAGE analysis of newly synthesized mitochondrial translation products showed an accumulation of free COXI. Quantitative mass spectrophotometry of an N-terminal COXI peptide showed that the ratio of formylated to unmodified N-termini in the assembled complex IV was ∼350:1 in controls and 4:1 in patient cells. These results show that mitochondrial protein synthesis can occur with inefficient formylation of methionyl-tRNA(Met), but that assembly of complex IV is impaired if the COXI N-terminus is not formylated.

The 3' addition of CCA to mitochondrial tRNASer(AGY) is specifically impaired in patients with mutations in the tRNA nucleotidyl transferase TRNT1.

Addition of the trinucleotide cytosine/cytosine/adenine (CCA) to the 3' end of transfer RNAs (tRNAs) is essential for translation and is catalyzed by the enzyme TRNT1 (tRNA nucleotidyl transferase), which functions in both the cytoplasm and mitochondria. Exome sequencing revealed TRNT1 mutations in two unrelated subjects with different clinical features. The first presented with acute lactic acidosis at 3 weeks of age and developed severe developmental delay, hypotonia, microcephaly, seizures, progressive cortical atrophy, neurosensorial deafness, sideroblastic anemia and renal Fanconi syndrome, dying at 21 months. The second presented at 3.5 years with gait ataxia, dysarthria, gross motor regression, hypotonia, ptosis and ophthalmoplegia and had abnormal signals in brainstem and dentate nucleus. In subject 1, muscle biopsy showed combined oxidative phosphorylation (OXPHOS) defects, but there was no OXPHOS deficiency in fibroblasts from either subject, despite a 10-fold-reduction in TRNT1 protein levels in fibroblasts of the first subject. Furthermore, in normal controls, TRNT1 protein levels are 10-fold lower in muscle than in fibroblasts. High resolution northern blots of subject fibroblast RNA suggested incomplete CCA addition to the non-canonical mitochondrial tRNA(Ser(AGY)), but no obvious qualitative differences in other mitochondrial or cytoplasmic tRNAs. Complete knockdown of TRNT1 in patient fibroblasts rendered mitochondrial tRNA(Ser(AGY)) undetectable, and markedly reduced mitochondrial translation, except polypeptides lacking Ser(AGY) codons. These data suggest that the clinical phenotypes associated with TRNT1 mutations are largely due to impaired mitochondrial translation, resulting from defective CCA addition to mitochondrial tRNA(Ser(AGY)), and that the severity of this biochemical phenotype determines the severity and tissue distribution of clinical features.

RMND1 deficiency associated with neonatal lactic acidosis, infantile onset renal failure, deafness, and multiorgan involvement.

RMND1 is an integral inner membrane mitochondrial protein that assembles into a large 240 kDa complex to support translation of the 13 polypeptides encoded on mtDNA, all of which are essential subunits of the oxidative phosphorylation (OXPHOS) complexes. Variants in RMND1 produce global defects in mitochondrial translation and were first reported in patients with severe neurological phenotypes leading to mortality in the first months of life. Using whole-exome sequencing, we identified compound heterozygous RMND1 variants in a 4-year-old patient with congenital lactic acidosis, severe myopathy, hearing loss, renal failure, and dysautonomia. The levels of mitochondrial ribosome proteins were reduced in patient fibroblasts, causing a translation defect, which was rescued by expression of the wild-type cDNA. RMND1 was almost undetectable by immunoblot analysis in patient muscle and fibroblasts. BN-PAGE analysis showed a severe combined OXPHOS assembly defect that was more prominent in patient muscle than in fibroblasts. Immunofluorescence experiments showed that RMND1 localizes to discrete foci in the mitochondrial network, juxtaposed to RNA granules where the primary mitochondrial transcripts are processed. RMND1 foci were not detected in patient fibroblasts. We hypothesize that RMND1 acts to anchor or stabilize the mitochondrial ribosome near the sites where the mRNAs are matured, spatially coupling post-transcriptional handling mRNAs with their translation, and that loss of function variants in RMND1 are associated with a unique constellation of clinical phenotypes that vary with the severity of the mitochondrial translation defect.

Tissue-specific responses to the LRPPRC founder mutation in French Canadian Leigh Syndrome.

French Canadian Leigh Syndrome (LSFC) is an early-onset, progressive neurodegenerative disorder with a distinct pattern of tissue involvement. Most cases are caused by a founder missense mutation in LRPPRC. LRPPRC forms a ribonucleoprotein complex with SLIRP, another RNA-binding protein, and this stabilizes polyadenylated mitochondrial mRNAs. LSFC fibroblasts have reduced levels of LRPPRC and a specific complex IV assembly defect; however, further depletion of mutant LRPPRC results in a complete failure to assemble a functional oxidative phosphorylation system, suggesting that LRPPRC levels determine the nature of the biochemical phenotype. We tested this hypothesis in cultured muscle cells and tissues from LSFC patients. LRPPRC levels were reduced in LSFC muscle cells, resulting in combined complex I and IV deficiencies. A similar combined deficiency was observed in skeletal muscle. Complex IV was only moderately reduced in LSFC heart, but was almost undetectable in liver. Both of these tissues showed elevated levels of complexes I and III. Despite the marked biochemical differences, the steady-state levels of LRPPRC and mitochondrial mRNAs were extremely low, LRPPRC was largely detergent-insoluble, and SLIRP was undetectable in all LSFC tissues. The level of the LRPPRC/SLIRP complex appeared much reduced in control tissues by the first dimension blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis compared with fibroblasts, and even by second dimension analysis it was virtually undetectable in control heart. These results point to tissue-specific pathways for the post-transcriptional handling of mitochondrial mRNAs and suggest that the biochemical defects in LSFC reflect the differential ability of tissues to adapt to the mutation.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations.

The conserved interaction of C7orf30 with MRPL14 promotes biogenesis of the mitochondrial large ribosomal subunit and mitochondrial translation.

Mammalian mitochondria harbor a dedicated translation apparatus that is required for the synthesis of 13 mitochondrial DNA (mtDNA)-encoded polypeptides, all of which are essential components of the oxidative phosphorylation (OXPHOS) complexes. Little is known about the mechanism of assembly of the mitoribosomes that catalyze this process. Here we show that C7orf30, a member of the large family of DUF143 proteins, associates with the mitochondrial large ribosomal subunit (mt-LSU). Knockdown of C7orf30 by short hairpin RNA (shRNA) does not alter the sedimentation profile of the mt-LSU, but results in the depletion of several mt-LSU proteins and decreased monosome formation. This leads to a mitochondrial translation defect, involving the majority of mitochondrial polypeptides, and a severe OXPHOS assembly defect. Immunoprecipitation and mass spectrometry analyses identified mitochondrial ribosomal protein (MRP)L14 as the specific interacting protein partner of C7orf30 in the mt-LSU. Reciprocal experiments in which MRPL14 was depleted by small interfering RNA (siRNA) phenocopied the C7orf30 knockdown. Members of the DUF143 family have been suggested to be universally conserved ribosomal silencing factors, acting by sterically inhibiting the association of the small and large ribosomal subunits. Our results demonstrate that, although the interaction between C7orf30 and MRPL14 has been evolutionarily conserved, human C7orf30 is, on the contrary, essential for mitochondrial ribosome biogenesis and mitochondrial translation.

Radioactive labeling of mitochondrial translation products in cultured cells.

The mammalian mitochondrial genome contains 37 genes, 13 of which encode polypeptide subunits in the enzyme complexes of the oxidative phosphorylation system. The other genes encode the rRNAs and tRNAs necessary for their translation. The mitochondrial translation machinery is located in the mitochondrial matrix, and is exclusively dedicated to the synthesis of these 13 enzyme subunits. Mitochondrial disease in humans is often associated with defects in mitochondrial translation. This can manifest as a global decrease in the rate of mitochondrial protein synthesis, a decrease in the synthesis of specific polypeptides, the synthesis of abnormal polypeptides, or in altered stability of specific translation products. All of these changes in the normal pattern of mitochondrial translation can be assessed by a straightforward technique that takes advantage of the insensitivity of the mitochondrial translation machinery to antibiotics that completely inhibit cytoplasmic translation. Thus, specific radioactive labeling of the mitochondrial translation products can be achieved in cultured cells, and the results can be visualized on gradient gels. The analysis of mitochondrial translation in cells cultured from patient biopsies is useful in the study of disease-causing mutations in both the mitochondrial and the nuclear genomes.

The 2-thiouridylase function of the human MTU1 (TRMU) enzyme is dispensable for mitochondrial translation.

MTU1 (TRMU) is a mitochondrial enzyme responsible for the 2-thiolation of the wobble U in tRNA(Lys), tRNA(Glu) and tRNA(Gln), a post-transcriptional modification believed to be important for accurate and efficient synthesis of the 13 respiratory chain subunits encoded by mtDNA. Mutations in MTU1 are associated with acute infantile liver failure, and this has been ascribed to a transient lack of cysteine, the sulfur donor for the thiouridylation reaction, resulting in a mitochondrial translation defect during early development. A mutation in tRNA(Lys) that causes myoclonic epilepsy with ragged-red fibers (MERRF) is also reported to prevent modification of the wobble U. Here we show that mitochondrial translation is unaffected in fibroblasts from an MTU1 patient, in which MTU1 is undetectable by immunoblotting, despite the severe reduction in the 2-thiolation of mitochondrial tRNA(Lys), tRNA(Glu) and tRNA(Gln). The only respiratory chain abnormality that we could observe in these cells was an accumulation of a Complex II assembly intermediate, which, however, did not affect the level of the fully assembled enzyme. The identical phenotype was observed by siRNA-mediated knockdown of MTU1 in HEK 293 cells. Further, the mitochondrial translation deficiencies present in myoblasts from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episode and MERRF patients, which are associated with defects in post-transcriptional modification of mitochondrial tRNAs, did not worsen following knockdown of MTU1 in these cells. This study demonstrates that MTU1 is not required for mitochondrial translation at normal steady-state levels of tRNAs, and that it may possess an as yet uncharacterized function in another sulfur-trafficking pathway.

LRPPRC and SLIRP interact in a ribonucleoprotein complex that regulates posttranscriptional gene expression in mitochondria.

Mutations in LRPPRC are responsible for the French Canadian variant of Leigh syndrome (LSFC), a neurodegenerative disorder caused by a tissue-specific deficiency in cytochrome c oxidase (COX). To investigate the pathogenic mechanism of disease, we studied LRPPRC function in LSFC and control fibroblasts. The level of mutated LRPPRC is reduced in LSFC cells, and this results in decreased steady-state levels of most mitochondrial mRNAs, but not rRNAs or tRNAs, a phenotype that can be reproduced by siRNA-mediated knockdown of LRPPRC in control cells. Processing of the primary transcripts appears normal. The resultant defect in mitochondrial protein synthesis in LSFC cells disproportionately affects the COX subunits, leading to an isolated COX assembly defect. Further knockdown of LRPPRC produces a generalized assembly defect in all oxidative phosphorylation complexes containing mtDNA-encoded subunits, due to a severe decrease in all mitochondrial mRNAs. LRPPRC exists in a high-molecular-weight complex, and it coimmunoprecipitates with SLIRP, a stem-loop RNA-binding protein. Although this interaction does not depend on mitochondrial mRNA, both proteins show reduced stability in its absence. These results implicate LRPPRC in posttranscriptional mitochondrial gene expression as part of a ribonucleoprotein complex that regulates the stability and handling of mature mRNAs.

Oxidative phosphorylation: synthesis of mitochondrially encoded proteins and assembly of individual structural subunits into functional holoenzyme complexes.

The bulk of ATP consumed by various cellular processes in higher eukaryotes is normally produced by five multimeric protein complexes (I-V) embedded within the inner mitochondrial membrane, in a process known as oxidative phosphorylation (OXPHOS). Maintenance of energy homeostasis under most physiological conditions is therefore contingent upon the ability of OXPHOS to meet cellular changes in bioenergetic demand, with a chronic failure to do so being a frequent cause of human disease. With the exception of Complex II, the structural subunits of OXPHOS complexes are encoded by both the nuclear and the mitochondrial genomes. The physical separation of the two genomes necessitates that the expression of the 13 mitochondrially encoded polypeptides be co-ordinated with that of relevant nuclear-encoded partners in order to assemble functional holoenzyme complexes. Complex biogenesis is a highly ordered process, and several nuclear-encoded factors that function at distinct stages in the assembly of individual OXPHOS complexes have been identified.

Human SCO2 is required for the synthesis of CO II and as a thiol-disulphide oxidoreductase for SCO1.

Human SCO1 and SCO2 code for essential metallochaperones with ill-defined functions in the biogenesis of the CuA site of cytochrome c oxidase subunit II (CO II). Here, we have used patient cell lines to investigate the specific roles of each SCO protein in this pathway. By pulse-labeling mitochondrial translation products, we demonstrate that the synthesis of CO II is reduced in SCO2, but not in SCO1, cells. Despite this biosynthetic defect, newly synthesized CO II is more stable in SCO2 cells than in control cells. RNAi-mediated knockdown of mutant SCO2 abolishes CO II labeling in the translation assay, whereas knockdown of mutant SCO1 does not affect CO II synthesis. These results indicate that SCO2 acts upstream of SCO1, and that it is indispensable for CO II synthesis. The subsequent maturation of CO II is contingent upon the formation of a complex that includes both SCO proteins, each with a functional CxxxC copper-coordinating motif. In control cells, the cysteines in this motif in SCO1 exist as a mixed population comprised of oxidized disulphides and reduced thiols; however, the relative ratio of oxidized to reduced cysteines in SCO1 is perturbed in cells from both SCO backgrounds. Overexpression of wild-type SCO2, or knockdown of mutant SCO2, in SCO2 cells alters the ratio of oxidized to reduced cysteines in SCO1, suggesting that SCO2 acts as a thiol-disulphide oxidoreductase to oxidize the copper-coordinating cysteines in SCO1 during CO II maturation. Based on these data we present a model in which each SCO protein fulfills distinct, stage-specific functions during CO II synthesis and CuA site maturation.

The A3243G tRNALeu(UUR) MELAS mutation causes amino acid misincorporation and a combined respiratory chain assembly defect partially suppressed by overexpression of EFTu and EFG2.

The majority of patients with MELAS (mitochondrial encephalomyophathy, lactic acidosis, stroke-like episodes) carry a heteroplasmic A3243G mutation in the mitochondrial tRNA(Leu(UUR)). The mutation prevents modification of the wobble U base, impairing translation at UUA and UUG codons; however, whether this results in amino acid misincorporation in the mitochondrial translation products remains controversial. We tested this hypothesis in homoplasmic mutant myoblasts isolated from a MELAS patient and investigated whether overexpression of the mitochondrial translation elongation factors could suppress the translation defect. Blue-Native gel electrophoretic analysis demonstrated an almost complete lack of assembly of respiratory chain complexes I, IV and V in MELAS myoblasts. This phenotype could be partially suppressed by overexpression of EFTu or EFG2 but not EFTs or EFG1. Despite the severity of the assembly defect, overall mitochondrial protein synthesis was only moderately affected, but some anomalously migrating translation products were present. Pulse-chase labeling showed reduced stability of all mitochondrial translation products consistent with the assembly defect. Labeling patterns of the translation products were similar with [(3)H]-leucine or [(3)H]-phenylalanine, showing that loss of the wobble U modification did not permit decoding of UUY codons; however, endoproteinase fingerprint analysis showed clear evidence of amino acid misincorporation in three polypeptides: CO III, CO II and ATP6. Taken together, these data demonstrate that the A3243G mutation produces both loss- and gain-of-function phenotypes, explaining the apparent discrepancy between the severity of the translation and respiratory chain assembly defects, and suggest a function for EFG2 in quality control of translation elongation.

Distinct clinical phenotypes associated with a mutation in the mitochondrial translation elongation factor EFTs.

The 13 polypeptides encoded in mitochondrial DNA (mtDNA) are synthesized in the mitochondrial matrix on a dedicated protein-translation apparatus that resembles that found in prokaryotes. Here, we have investigated the genetic basis for a mitochondrial protein-synthesis defect associated with a combined oxidative phosphorylation enzyme deficiency in two patients, one of whom presented with encephalomyopathy and the other with hypertrophic cardiomyopathy. Sequencing of candidate genes revealed the same homozygous mutation (C997T) in both patients in TSFM, a gene coding for the mitochondrial translation elongation factor EFTs. EFTs functions as a guanine nucleotide exchange factor for EFTu, another translation elongation factor that brings aminoacylated transfer RNAs to the ribosomal A site as a ternary complex with guanosine triphosphate. The mutation predicts an Arg333Trp substitution at an evolutionarily conserved site in a subdomain of EFTs that interacts with EFTu. Molecular modeling showed that the substitution disrupts local subdomain structure and the dimerization interface. The steady-state levels of EFTs and EFTu in patient fibroblasts were reduced by 75% and 60%, respectively, and the amounts of assembled complexes I, IV, and V were reduced by 35%-91% compared with the amounts in controls. These phenotypes and the translation defect were rescued by retroviral expression of either EFTs or EFTu. These data clearly establish mutant EFTs as the cause of disease in these patients. The fact that the same mutation is associated with distinct clinical phenotypes suggests the presence of genetic modifiers of the mitochondrial translation apparatus.