Targeting of the cytosolic poly(A) binding protein PABPC1 to mitochondria causes mitochondrial translation inhibition

Mammalian mitochondria contain their own genome that is almost fully transcribed from both strands, generating polycistronic RNA units that are processed and matured. The mitochondrial mRNA is modified by oligo- or polyadenylation at the 3′ termini, but the exact function of this post-transcriptional addition is unclear. Current debate focuses on the role of polyadenylation in transcript stability. An equally likely function that has received little attention is that, as in the cytosol of eukaryotes, polyadenylation facilitates translation in the mitochondrion. To address this issue, we have targeted cytosolic proteins to the mitochondrion, a poly(A) specific 3′ exoribonuclease, mtPARN, and a poly(A)binding protein, mtPABP1. Removal of the 3′ adenylyl extensions had a variable effect on mt-mRNA steady-state levels, increasing (MTND1, 2, 5) or decreasing (MTCO1, 2, RNA14) certain species with minimal effect on others (RNA7, MTND3). Translation was markedly affected, but interpretation of this was complicated by the concomitant 3′ truncation of the open reading frame in most cases. Coating of the poly(A) tail by mtPABP1, however, did not lead to transcript decay but caused a marked inhibition of mitochondrial translation. These data are consistent with endogenous RNA-binding factor(s) interacting with the poly(A) to optimize mitochondrial protein synthesis

ERAL1 is associated with mitochondrial ribosome and elimination of ERAL1 leads to mitochondrial dysfunction and growth retardation

ERAL1, a homologue of Era protein in Escherichia coli, is a member of conserved GTP-binding proteins with RNA-binding activity. Depletion of prokaryotic Era inhibits cell division without affecting chromosome segregation. Previously, we isolated ERAL1 protein as one of proteins which were associated with mitochondrial transcription factor A by using immunoprecipitation. In this study, we analysed the localization and function of ERAL1 in mammalian cells. ERAL1 was localized in mitochondrial matrix and associated with mitoribosomal proteins including the 12S rRNA. siRNA knockdown of ERAL1 decreased mitochondrial translation, caused redistribution of ribosomal small subunits and reduced 12S rRNA. The knockdown of ERAL1 in human HeLa cells elevated mitochondrial superoxide production and slightly decreased mitochondrial membrane potential. The knockdown inhibited the growth of HeLa cells with an accumulation of apoptotic cells. These results suggest that ERAL1 is localized in a small subunit of the mitochondrial ribosome, plays an important role in the small ribosomal constitution, and is also involved in cell viability

Identification and characterization of novel genes involved in cytochrome c oxidase deficiencies

In mitochondria, ATP is generated by oxidative phosphorylation (OXPHOS), a process that requires five multimeric enzyme complexes. Electrons are passed along the first four enzyme complexes (complex I-IV) that make up the mitochondria respiratory chain, releasing energy that is stored in the form of a proton gradient across the mitochondrial inner membrane, and is subsequently used by the ATP synthase (complex V) to produce ATP. Complex IV or cytochrome c oxidase (COX) is the terminal enzyme in the mitochondrial respiratory chain, catalyzing the oxidation of cytochrome c by molecular oxygen. It contains 13 structural subunits in mammals, 3 of which are encoded by mitochondrial DNA. Cytochrome c oxidase deficiencies can be caused by mutations in either mitochondrial or nuclear DNA. COX deficiency can result from mutations in the structural subunits or factors necessary for the assembly of the enzyme complex. In this thesis, two novel genes mutated in two subjects with COX deficiency have been identified. First, we identified a specific defect in the synthesis of the mtDNA-encoded COX subunit 1 (COX I) in a pedigree segregating late-onset Leigh Syndrome and COX deficiency. We mapped the defect to chromosome 17q by microcell-mediated chromosome transfer and identified a homozygous single base pair insertion causing a premature stop in CCDC44, renamed TACO1 for translational activator of COX I. TACO1 is a member of a large family of hypothetical proteins containing a conserved DUF28 domain that localizes to the mitochondrial matrix. Expression of the wild-type cDNA restored TACO1 protein and rescued the translation defect. TACO1 is the first specific mitochondrial translational activator identified in mammals. Respiratory competence, mitochondrial translation and COX activity were normal in yeast strain deleted for the orthologue YGR021w, suggesting that TACO1 has evolved a novel function in mammalian mitochondrial translation. Secondly, we studied a family in which the subject presented with severe congenital lactic acidosis and dysmorphic features associated with a COX assembly defect and a specific decrease in the synthesis of COX I. Using a combination of microcell mediated chromosome transfer, homozygosity mapping, and transcript profiling we mapped the gene defect to chromosome 12, and identified a homozygous missense mutation causing an amino acid change from methionine to isoleucine in C12orf62, a gene apparently restricted to the vertebrate lineage. Expression of the wild-type cDNA restored C12orf62 protein levels, and rescued the COX I synthesis and COX assembly defect. C12orf62 is a very small (6 kDa), uncharacterized, single transmembrane protein that localizes to mitochondria. COX I, II and IV subunits co-immunoprecipitated with an epitope-tagged version of C12orf62, and 2D BN-PAGE analysis of newly synthesized mitochondrial COX subunits in subject fibroblasts showed that COX assembly was impaired, and the nascent enzyme complex unstable. We conclude that C12orf62 is required for coordinating the early steps of COX assembly with the synthesis of COX I.Dans les mitochondries, l'ATP est généré par la phosphorylation oxydative (PHOSOX), un processus qui nécessite cinq complexes enzymatiques multimériques. Le transport des électrons le long des quatre premiers complexes enzymatiques (complexes I-IV) libère l'énergie qui est stockée sous la forme d'un gradient de protons à travers la membrane interne mitochondriale et est ensuite utilisée par l'ATP synthétase (complexe V) pour produire de l'ATP. Le complexe IV ou cytochrome C oxydase (COX) est l'enzyme terminale de la chaîne respiratoire mitochondriale, catalysant l'oxydation du cytochrome c par l'oxygène moléculaire. Il contient 13 sous-unités structurelles chez les mammifères, dont 3 sont codées par l'ADN mitochondriale. Les déficiences en cytochrome C oxydase peuvent être causées par des mutations dans l'ADN mitochondriale ou l'ADN nucléaire. Les carences en COX peuvent être liées à des mutations dans les sous-unités structurelles ou à des mutations dans des facteurs nécessaires à l'assemblage du complexe enzymatique. Dans cette thèse, deux nouveaux gènes mutés ont été identifiés et caractérisés dans deux patients présentant un déficit en COX. Premièrement, nous avons identifié un défaut spécifique dans la synthèse de la sous-unité COX 1 de l'ADN mitochondriale (COX I) dans un pedigree présentant une apparition tardive du syndrome de Leigh et une carence en COX. Nous avons cartographié le défaut génétique au chromosome 17q par la technique de transfert de chromosomes à médiation microcellulaire. Nous avons, par la suite, identifié une mutation homozygote, une insertion d'une base causant l'apparition prématurée d'un codon stop qui entraîne l'arrêt de la synthèse de la protéine CCDC44, renommé TACO1 pour activateur de la traduction de la COX I. TACO1 est membre d'une famille de protéines contenant un domaine conservé à fonction inconnue, nommé DUF28, qui se localise à la matrice mitochondriale. L'expression de l'ADN complémentaire de type sauvage de TACO1 compense le défaut de traduction de COX I. TACO1 est le premier activateur spécifique de la traduction mitochondriale à être identifié chez les mammifères. Il a été observé que l'absence (knock-down) du gène codant l'orthologue de TACO1 chez la levure, le YGR021w, ne perturbait pas la compétence des voies respiratoires, la traduction mitochondriale, ni l'activité de COX. Ceci suggère que TACO1 a évolué et a acquérit une nouvelle fonction dans la traduction mitochondriale chez les mammifères. Deuxièmement, nous avons étudié une famille dans laquelle le sujet présentait une acidose lactique congénitale et dysmorphie associée à un défaut d'assemblage et une diminution de l'activité enzymatique de la COX due à un défaut spécifique dans la traduction de COX I. En utilisant une combinaison de techniques dont le transfert de chromosomes à médiation microcellulaire, la cartographie d'homozygotie et le profilage de transcription, nous avons cartographié le gène défectueux sur le chromosome 12. Nous avons identifié une mutation faux sens à l'état homozygote provoquant un changement d'acide aminé de méthionine en isoleucine dans le gène C12orf62, un gène qui semble restreint à la lignée des vertébrés. L'expression de l'ADN complémentaire de type sauvage de C12orf62 a restauré la synthèse de COX I et le défaut d'assemblage de la COX. C12orf62 est une très petite protéine transmembranaire (6 kDa), non caractérisée, qui se localise aux mitochondries. Les sous-unités COX I, II et IV co-immunoprécipitent avec un épitope marqué de la protéine C12orf62. Les analyses de bleu d'électrophorèse sur gel de polyacrylamide natif (BN-PAGE) en deux dimensions pour les sous-unités nouvellement synthétisées de la COX mitochondriale ont démontré que la COX assemblée est altérée et que le complexe enzymatique naissant est instable dans les fibroblastes du patient atteint. Nous concluons que C12orf62 est nécessaire pour coordonner les étapes précoces de l'assemblage de la COX et de la synthèse de COX I

Stomatin-like protein 2 deficiency results in impaired mitochondrial translation

Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATPsynthase complexes that are essential for the assembly and function of these complexes.This process occurs in close proximity to the mitochondrial inner membrane. However, themechanisms and molecular machinery involved in mitochondrial translation are not fullyunderstood, and defects in this process can result in severe diseases. Stomatin-like protein(SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enrichedmicrodomains in the mitochondrial inner membrane that are important for the formation ofrespiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processesclosely associated with the mitochondrial inner membrane, we hypothesized that thefunction of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteinepulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout miceshowed a significant impairment in the production of several mitochondrial DNA-encodedpolypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6.Measurement of mitochondrial DNA stability and mitochondrial transcription revealed thatthis impairment was at the post-transcriptional level. [...

Stomatin-like protein 2 deficiency results in impaired mitochondrial translation.

Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATP synthase complexes that are essential for the assembly and function of these complexes. This process occurs in close proximity to the mitochondrial inner membrane. However, the mechanisms and molecular machinery involved in mitochondrial translation are not fully understood, and defects in this process can result in severe diseases. Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enriched microdomains in the mitochondrial inner membrane that are important for the formation of respiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processes closely associated with the mitochondrial inner membrane, we hypothesized that the function of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteine pulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout mice showed a significant impairment in the production of several mitochondrial DNA-encoded polypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6. Measurement of mitochondrial DNA stability and mitochondrial transcription revealed that this impairment was at the post-transcriptional level. Examination of mitochondrial ribosome assembly showed that SLP-2 migrated in sucrose-density gradients similarly to the large ribosomal subunit but that its deletion at the genetic level did not affect mitochondrial ribosome assembly. Functionally, the impairment in mitochondrial translation correlated with decreased interleukin-2 production in activated T cells. Altogether, these data show that SLP-2 acts as a general regulator of mitochondrial translation

MITRAC7 Acts as a COX1-Specific Chaperone and Reveals a Checkpoint during Cytochrome c Oxidase Assembly.

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, is assembled from mitochondria- and nuclear-encoded subunits. The MITRAC complex represents the central assembly intermediate during this process as it receives imported subunits and regulates mitochondrial translation of COX1 mRNA. The molecular processes that promote and regulate the progression of assembly downstream of MITRAC are still unknown. Here, we identify MITRAC7 as a constituent of a late form of MITRAC and as a COX1-specific chaperone. MITRAC7 is required for cytochrome c oxidase biogenesis. Surprisingly, loss of MITRAC7 or an increase in its amount causes selective cytochrome c oxidase deficiency in human cells. We demonstrate that increased MITRAC7 levels stabilize and trap COX1 in MITRAC, blocking progression in the assembly process. In contrast, MITRAC7 deficiency leads to turnover of newly synthesized COX1. Accordingly, MITRAC7 affects the biogenesis pathway by stabilizing newly synthesized COX1 in assembly intermediates, concomitantly preventing turnover.Open-Access Publikationsfonds 2015peerReviewe

Mitochondrial translation is impaired in SLP-2-deficient T cells upon stimulation.

<p>Resting or anti-CD3/CD28-stimulated T cells isolated from WT or SLP-2 T-KO mice were cultured with ³⁵S-methionine/cysteine for 1 h in the presence of the cytoplasmic translation inhibitor, emetine. Cell lysates were resolved by SDS-PAGE and ³⁵S-methionine/cysteine incorporation into nascent mitochondria-encoded polypeptides was measured by direct autoradiography. (A) Representative ³⁵S-methionine/cysteine incorporation data are shown. Bands corresponding to mitochondrial polypeptides are denoted. (B) Densitometric analysis of ³⁵S-methionine/cysteine incorporation from multiple experiments was performed with all values relative to the WT Stimulated group, which was assigned a value of 1 (represented by dotted line). Values below dotted line indicate decreased densitometry relative to WT Stimulated group. N = 4 (resting) or 7 (stimulated) mice per group. Statistical significance was calculated using Student's t test. *: p < 0.05; **: p < 0.01; ***: p < 0.001 compared to WT Stimulated group.</p