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The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mutations in the nuclear-encoded mitochondrial gene CHCHD10 have been observed in patients with a spectrum of diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathogenic nature of disease-associated variants of CHCHD10 we generated a zebrafish knock-in (KI) model expressing the orthologous ALS-associated CHCHD10 P80L variant (zebrafish: Chchd10 P83L ). Larval chchd10 P83L/P83L fish displayed reduced Chchd10 protein expression levels, motor impairment, reduced survival and abnormal neuromuscular junctions (NMJ). These deficits were not accompanied by changes in transcripts involved in the integrated stress response (ISR), phenocopying previous findings in our knockout (chchd10 -/- ). Adult, 11-month old chchd10 P83L/P83L zebrafish, displayed smaller slow- and fast-twitch muscle cell cross-sectional areas compared to wild type zebrafish muscle cells. Motoneurons in the spinal cord of chchd10 P83L/P83L zebrafish displayed similar cross-sectional areas to that of wild type motor neurons and significantly fewer motor neurons were observed when compared to chchd2 -/- adult spinal cords. Bulk RNA sequencing using whole spinal cords of 7-month old fish revealed transcriptional changes associated with neuroinflammation, apoptosis, amino acid metabolism and mt-DNA inflammatory response in our chchd10 P83L/P83L model. The findings presented here, suggest that the CHCHD10 P80L variant confers an ALS-like phenotype when expressed in zebrafish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The human mitochondrial ribosome contains three [2Fe-2S] clusters whose assembly pathway, role, and implications for mitochondrial and metabolic diseases are unknown. Here, structure-function correlation studies show that the clusters play a structural role during mitoribosome assembly. To uncover the assembly pathway, we have examined the effect of silencing the expression of Fe-S cluster biosynthetic and delivery factors on mitoribosome stability. We find that the mitoribosome receives its [2Fe-2S] clusters from the GLRX5-BOLA3 node. Additionally, the assembly of the small subunit depends on the mitoribosome biogenesis factor METTL17, recently reported containing a [4Fe-4S] cluster, which we propose is inserted via the ISCA1-NFU1 node. Consistently, fibroblasts from subjects suffering from 'multiple mitochondrial dysfunction' syndrome due to mutations in BOLA3 or NFU1 display previously unrecognized attenuation of mitochondrial protein synthesis that contributes to their cellular and pathophysiological phenotypes. Finally, we report that, in addition to their structural role, one of the mitoribosomal [2Fe-2S] clusters and the [4Fe-4S] cluster in mitoribosome assembly factor METTL17 sense changes in the redox environment, thus providing a way to regulate organellar protein synthesis accordingly., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mitochondria interact with the ER at structurally and functionally specialized membrane contact sites known as mitochondria-ER contact sites (MERCs). Combining proximity labelling (BioID), co-immunoprecipitation, confocal microscopy and subcellular fractionation, we found that the ER resident SMP-domain protein ESYT1 was enriched at MERCs, where it forms a complex with the outer mitochondrial membrane protein SYNJ2BP. BioID analyses using ER-targeted, outer mitochondrial membrane-targeted, and MERC-targeted baits, confirmed the presence of this complex at MERCs and the specificity of the interaction. Deletion of ESYT1 or SYNJ2BP reduced the number and length of MERCs. Loss of the ESYT1-SYNJ2BP complex impaired ER to mitochondria calcium flux and provoked a significant alteration of the mitochondrial lipidome, most prominently a reduction of cardiolipins and phosphatidylethanolamines. Both phenotypes were rescued by reexpression of WT ESYT1 and an artificial mitochondria-ER tether. Together, these results reveal a novel function for ESYT1 in mitochondrial and cellular homeostasis through its role in the regulation of MERCs., (© 2023 Janer et al.)

Mutations in SLC25A46 underlie a wide spectrum of neurodegenerative diseases associated with alterations in mitochondrial morphology. We established an SLC25A46 knock-out cell line in human fibroblasts and studied the pathogenicity of three variants (p.T142I, p.R257Q, and p.E335D). Mitochondria were fragmented in the knock-out cell line and hyperfused in all pathogenic variants. The loss of SLC25A46 led to abnormalities in the mitochondrial cristae ultrastructure that were not rescued by the expression of the variants. SLC25A46 was present in discrete puncta at mitochondrial branch points and tips of mitochondrial tubules, co-localizing with DRP1 and OPA1. Virtually, all fission/fusion events were demarcated by a SLC25A46 focus. SLC25A46 co-immunoprecipitated with the fusion machinery, and loss of function altered the oligomerization state of OPA1 and MFN2. Proximity interaction mapping identified components of the ER membrane, lipid transfer proteins, and mitochondrial outer membrane proteins, indicating that it is present at interorganellar contact sites. SLC25A46 loss of function led to altered mitochondrial lipid composition, suggesting that it may facilitate interorganellar lipid flux or play a role in membrane remodeling associated with mitochondrial fusion and fission., (© 2023 Schuettpelz et al.)

Mutations in CHCHD10 and CHCHD2, encoding two paralogous mitochondrial proteins, have been identified in cases of amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Parkinson's disease. Their role in disease is unclear, though both have been linked to mitochondrial respiration and mitochondrial stress responses. Here, we investigated the biological roles of these proteins during vertebrate development using knockout (KO) models in zebrafish. We demonstrate that loss of either or both proteins leads to motor impairment, reduced survival and compromised neuromuscular junction integrity in larval zebrafish. Compensation by Chchd10 was observed in the chchd2 -/- model, but not by Chchd2 in the chchd10 -/- model. The assembly of mitochondrial respiratory chain Complex I was impaired in chchd10 -/- and chchd2 -/- zebrafish larvae, but unexpectedly not in a double chchd10 -/- and chchd2 -/- model, suggesting that reduced mitochondrial Complex I cannot be solely responsible for the observed phenotypes, which are generally more severe in the double KO. We observed transcriptional activation markers of the mitochondrial integrated stress response (mt-ISR) in the double chchd10 -/- and chchd2 -/- KO model, suggesting that this pathway is involved in the restoration of Complex I assembly in our double KO model. The data presented here demonstrates that the Complex I assembly defect in our single KO models arises independently of the mt-ISR. Furthermore, this study provides evidence that both proteins are required for normal vertebrate development., (© 2023 The Authors. Developmental Neurobiology published by Wiley Periodicals LLC.)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons. Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT), which catalyzes the first step in the de novo synthesis of sphingolipids (SLs), cause childhood-onset ALS. SPTLC1-ALS variants map to a transmembrane domain that interacts with ORMDL proteins, negative regulators of SPT activity. We show that ORMDL binding to the holoenzyme complex is impaired in cells expressing pathogenic SPTLC1-ALS alleles, resulting in increased SL synthesis and a distinct lipid signature. C-terminal SPTLC1 variants cause peripheral hereditary sensory and autonomic neuropathy type 1 (HSAN1) due to the synthesis of 1-deoxysphingolipids (1-deoxySLs) that form when SPT metabolizes L-alanine instead of L-serine. Limiting L-serine availability in SPTLC1-ALS-expressing cells increased 1-deoxySL and shifted the SL profile from an ALS to an HSAN1-like signature. This effect was corroborated in an SPTLC1-ALS pedigree in which the index patient uniquely presented with an HSAN1 phenotype, increased 1-deoxySL levels, and an L-serine deficiency. These data demonstrate how pathogenic variants in different domains of SPTLC1 give rise to distinct clinical presentations that are nonetheless modifiable by substrate availability.

With more than 40 causative genes identified so far, autosomal dominant cerebellar ataxias exhibit a remarkable genetic heterogeneity. Yet, half the patients are lacking a molecular diagnosis. In a large family with nine sampled affected members, we performed exome sequencing combined with whole-genome linkage analysis. We identified a missense variant in NPTX1, NM_002522.3:c.1165G>A: p.G389R, segregating with the phenotype. Further investigations with whole-exome sequencing and an amplicon-based panel identified four additional unrelated families segregating the same variant, for whom a common founder effect could be excluded. A second missense variant, NM_002522.3:c.980A>G: p.E327G, was identified in a fifth familial case. The NPTX1-associated phenotype consists of a late-onset, slowly progressive, cerebellar ataxia, with downbeat nystagmus, cognitive impairment reminiscent of cerebellar cognitive affective syndrome, myoclonic tremor and mild cerebellar vermian atrophy on brain imaging. NPTX1 encodes the neuronal pentraxin 1, a secreted protein with various cellular and synaptic functions. Both variants affect conserved amino acid residues and are extremely rare or absent from public databases. In COS7 cells, overexpression of both neuronal pentraxin 1 variants altered endoplasmic reticulum morphology and induced ATF6-mediated endoplasmic reticulum stress, associated with cytotoxicity. In addition, the p.E327G variant abolished neuronal pentraxin 1 secretion, as well as its capacity to form a high molecular weight complex with the wild-type protein. Co-immunoprecipitation experiments coupled with mass spectrometry analysis demonstrated abnormal interactions of this variant with the cytoskeleton. In agreement with these observations, in silico modelling of the neuronal pentraxin 1 complex evidenced a destabilizing effect for the p.E327G substitution, located at the interface between monomers. On the contrary, the p.G389 residue, located at the protein surface, had no predictable effect on the complex stability. Our results establish NPTX1 as a new causative gene in autosomal dominant cerebellar ataxias. We suggest that variants in NPTX1 can lead to cerebellar ataxia due to endoplasmic reticulum stress, mediated by ATF6, and associated to a destabilization of NP1 polymers in a dominant-negative manner for one of the variants., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The accumulation of sphingolipid species in the cell contributes to the development of obesity and neurological disease. However, the subcellular localization of sphingolipid-synthesizing enzymes is unclear, limiting the understanding of where and how these lipids accumulate inside the cell and why they are toxic. Here, we show that SPTLC2, a subunit of the serine palmitoyltransferase (SPT) complex, catalyzing the first step in de novo sphingolipid synthesis, localizes dually to the ER and the outer mitochondrial membrane. We demonstrate that mitochondrial SPTLC2 interacts and forms a complex in trans with the ER-localized SPT subunit SPTLC1. Loss of SPTLC2 prevents the synthesis of mitochondrial sphingolipids and protects from palmitate-induced mitochondrial toxicity, a process dependent on mitochondrial ceramides. Our results reveal the in trans assembly of an enzymatic complex at an organellar membrane contact site, providing novel insight into the localization of sphingolipid synthesis and the composition and function of ER-mitochondria contact sites., (© 2021 Aaltonen et al.)

Compartmentalization is a defining characteristic of eukaryotic cells, and partitions distinct biochemical processes into discrete subcellular locations. Microscopy 1 and biochemical fractionation coupled with mass spectrometry 2-4 have defined the proteomes of a variety of different organelles, but many intracellular compartments have remained refractory to such approaches. Proximity-dependent biotinylation techniques such as BioID provide an alternative approach to define the composition of cellular compartments in living cells 5-7 . Here we present a BioID-based map of a human cell on the basis of 192 subcellular markers, and define the intracellular locations of 4,145 unique proteins in HEK293 cells. Our localization predictions exceed the specificity of previous approaches, and enabled the discovery of proteins at the interface between the mitochondrial outer membrane and the endoplasmic reticulum that are crucial for mitochondrial homeostasis. On the basis of this dataset, we created humancellmap.org as a community resource that provides online tools for localization analysis of user BioID data, and demonstrate how this resource can be used to understand BioID results better.

Mutations in CHCHD10, coding for a mitochondrial intermembrane space protein, are a rare cause of autosomal dominant amyotrophic lateral sclerosis. Mutation-specific toxic gain of function or haploinsufficiency models have been proposed to explain pathogenicity. To decipher the metabolic dysfunction associated with the haploinsufficient p.R15L variant, we integrated transcriptomic, metabolomic and proteomic data sets in patient cells subjected to an energetic stress that forces the cells to rely on oxidative phosphorylation for ATP production. Patient cells had a complex I deficiency that resulted in an increased NADH/NAD+ ratio, diminished TCA cycle activity, a reorganization of one carbon metabolism and an increased AMP/ATP ratio leading to phosphorylation of AMPK and inhibition of mTORC1. These metabolic changes activated the unfolded protein response (UPR) in the ER through the IRE1/XBP1 pathway, upregulating downstream targets including ATF3, ATF4, CHOP and EGLN3, and two cytokine markers of mitochondrial disease, GDF15 and FGF21. Activation of the mitochondrial UPR was mediated through an upregulation of the transcription factors ATF4 and ATF5, leading to increased expression of mitochondrial proteases and heat shock proteins. There was a striking transcriptional up regulation of at least seven dual specific phosphatases, associated with an almost complete dephosphorylation of JNK isoforms, suggesting a concerted deactivation of MAP kinase pathways. This study demonstrates that loss of CHCHD10 function elicits an energy deficit that activates unique responses to nutrient stress in both the mitochondria and ER, which may contribute to the selective vulnerability of motor neurons., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The advent of whole-exome sequencing ushered in a new era of in the genetic diagnosis of rare diseases, but characterizing large alterations in genome architecture has remained challenging. In this issue of Med, Frazier et al. harnessed the power of genomics and proteomics to identify a recurrent duplication as the molecular basis of a fatal perinatal mitochondrial cardiomyopathy. 1 ., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mutations in LRPPRC cause Leigh Syndrome French Canadian (LSFC), an early onset neurodegenerative disease, with differential tissue involvement. The molecular basis for tissue specificity in this disease remains unknown. LRPPRC, an RNA binding protein, forms a stable complex with SLIRP, which binds to, and stabilizes mitochondrial mRNAs. In cell culture and animal models, loss of LRPPRC function results in transcript-specific alterations in the steady-state levels of mitochondrial mRNAs and poly (A) tail length, the mechanisms for which are not understood. The poly (A) tail length of mitochondrial mRNAs has not been investigated in human tissues from heathy subjects or LSFC patients. Here we have mapped the 3'-termini of mature mitochondrial mRNAs in three tissues (skeletal muscle, heart, and liver) from a healthy individual and an LSFC patient. We show that the poly (A) tail length of mitochondrial mRNAs varies amongst tissues, and that the missense mutation in LRPPRC that causes LSFC results in tissue- and transcript-specific deadenylation of a subset of mitochondrial mRNAs, likely contributing the nature and severity of the biochemical phenotype in different tissues. We also found a relatively large fraction of short transcripts lacking a stop codon, some with short poly (A) tails, in patient tissue, suggesting that mutations in LRPPRC may also impair proper 3' end processing of some mRNAs., (© 2020 The Authors.)

We used BioID, a proximity-dependent biotinylation assay with 100 mitochondrial baits from all mitochondrial sub-compartments, to create a high-resolution human mitochondrial proximity interaction network. We identified 1,465 proteins, producing 15,626 unique high-confidence proximity interactions. Of these, 528 proteins were previously annotated as mitochondrial, nearly half of the mitochondrial proteome defined by Mitocarta 2.0. Bait-bait analysis showed a clear separation of mitochondrial compartments, and correlation analysis among preys across all baits allowed us to identify functional clusters involved in diverse mitochondrial functions and to assign uncharacterized proteins to specific modules. We demonstrate that this analysis can assign isoforms of the same mitochondrial protein to different mitochondrial sub-compartments and show that some proteins may have multiple cellular locations. Outer membrane baits showed specific proximity interactions with cytosolic proteins and proteins in other organellar membranes, suggesting specialization of proteins responsible for contact site formation between mitochondria and individual organelles., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Biogenesis of mammalian mitochondrial ribosomes (mitoribosomes) involves several conserved small GTPases. Here, we report that the Obg family protein GTPBP5 or MTG2 is a mitochondrial protein whose absence in a TALEN-induced HEK293T knockout (KO) cell line leads to severely decreased levels of the 55S monosome and attenuated mitochondrial protein synthesis. We show that a fraction of GTPBP5 co-sediments with the large mitoribosome subunit (mtLSU), and crosslinks specifically with the 16S rRNA, and several mtLSU proteins and assembly factors. Notably, the latter group includes MTERF4, involved in monosome assembly, and MRM2, the methyltransferase that catalyzes the modification of the 16S mt-rRNA A-loop U1369 residue. The GTPBP5 interaction with MRM2 was also detected using the proximity-dependent biotinylation (BioID) assay. In GTPBP5-KO mitochondria, the mtLSU lacks bL36m, accumulates an excess of the assembly factors MTG1, GTPBP10, MALSU1 and MTERF4, and contains hypomethylated 16S rRNA. We propose that GTPBP5 primarily fuels proper mtLSU maturation by securing efficient methylation of two 16S rRNA residues, and ultimately serves to coordinate subunit joining through the release of late-stage mtLSU assembly factors. In this way, GTPBP5 provides an ultimate quality control checkpoint function during mtLSU assembly that minimizes premature subunit joining to ensure the assembly of the mature 55S monosome., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Advances in genomics have transformed our ability to identify the genetic causes of rare diseases (RDs), yet we have a limited understanding of the mechanistic roles of most genes in health and disease. When a novel RD gene is first discovered, there is minimal insight into its biological function, the pathogenic mechanisms of disease-causing variants, and how therapy might be approached. To address this gap, the Canadian Rare Diseases Models and Mechanisms (RDMM) Network was established to connect clinicians discovering new disease genes with Canadian scientists able to study equivalent genes and pathways in model organisms (MOs). The Network is built around a registry of more than 500 Canadian MO scientists, representing expertise for over 7,500 human genes. RDMM uses a committee process to identify and evaluate clinician-MO scientist collaborations and approve 25,000 Canadian dollars in catalyst funding. To date, we have made 85 clinician-MO scientist connections and funded 105 projects. These collaborations help confirm variant pathogenicity and unravel the molecular mechanisms of RD, and also test novel therapies and lead to long-term collaborations. To expand the impact and reach of this model, we made the RDMM Registry open-source, portable, and customizable, and we freely share our committee structures and processes. We are currently working with emerging networks in Europe, Australia, and Japan to link international RDMM networks and registries and enable matches across borders. We will continue to create meaningful collaborations, generate knowledge, and advance RD research locally and globally for the benefit of patients and families living with RD., Competing Interests: Declarations of Interest The authors declare no competing interests., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

LONP1, an AAA+ mitochondrial protease, is implicated in protein quality control, but its precise role in this process remains poorly understood. In this study, we have investigated the role of human LONP1 in mitochondrial proteostasis and gene expression. Depletion of LONP1 resulted in partial loss of mitochondrial DNA (mtDNA) and a complete suppression of mitochondrial translation associated with impaired ribosome biogenesis. The levels of a distinct subset of mitochondrial matrix proteins (SSBP1, MTERFD3, FASTKD2, and CLPX) increased in the presence of a catalytically dead form of LONP1, suggesting that they are bona fide LONP1 substrates. Unexpectedly, the unprocessed forms of the same proteins also accumulated in an insoluble protein fraction. This subset of unprocessed matrix proteins (but not their mature forms) accumulated following depletion of the mitochondrial processing peptidase MPP, though all other MPP substrates investigated were processed normally. Prolonged depletion of LONP1 produced massive matrix protein aggregates, robustly activated the integrated stress response (ISR) pathway, and resulted in stabilization of PINK1, a mitophagy marker. These results demonstrate that LONP1 and MPPαβ are together required for the maturation of a subset of LONP1 client proteins and that LONP1 activity is essential for the maintenance of mitochondrial proteostasis and gene expression., (Copyright © 2018 American Society for Microbiology.)

Post-transcriptional RNA modifications play a critical role in the pathogenesis of human mitochondrial disorders, but the mechanisms by which specific modifications affect mitochondrial protein synthesis remain poorly understood. Here we used a quantitative RNA sequencing approach to investigate, at nucleotide resolution, the stoichiometry and methyl modifications of the entire mitochondrial tRNA pool, and establish the relevance to human disease. We discovered that a N 1 -methyladenosine (m 1 A) modification is missing at position 58 in the mitochondrial tRNA Lys of patients with the mitochondrial DNA mutation m.8344 A > G associated with MERRF (myoclonus epilepsy, ragged-red fibers). By restoring the modification on the mitochondrial tRNA Lys , we demonstrated the importance of the m 1 A58 to translation elongation and the stability of selected nascent chains. Our data indicates regulation of post-transcriptional modifications on mitochondrial tRNAs is finely tuned for the control of mitochondrial gene expression. Collectively, our findings provide novel insight into the regulation of mitochondrial tRNAs and reveal greater complexity to the molecular pathogenesis of MERRF.

Coiled-helix coiled-helix domain containing protein 10 (CHCHD10) and its paralogue CHCHD2 belong to a family of twin CX9C motif proteins, most of which localize to the intermembrane space of mitochondria. Dominant mutations in CHCHD10 cause amyotrophic lateral sclerosis (ALS)/frontotemporal dementia, and mutations in CHCHD2 have been associated with Parkinson's disease, but the function of these proteins remains unknown. Here we show that the p.R15L CHCHD10 variant in ALS patient fibroblasts destabilizes the protein, leading to a defect in the assembly of Complex I, impaired cellular respiration, mitochondrial hyperfusion, an increase in the steady-state level of CHCHD2, and a severe proliferation defect on galactose, a substrate that forces cells to synthesize virtually all of their ATP aerobically. CHCHD10 and CHCHD2 appeared together in distinct foci by immunofluorescence analysis and could be quantitatively immunoprecipitated with antibodies against either protein. Blue native polyacrylamide gel electrophoresis analyses showed that both proteins migrated in a high molecular weight complex (220 kDa) in control cells, which was, however, absent in patient cells. CHCHD10 and CHCHD2 levels increased markedly in control cells in galactose medium, a response that was dampened in patient cells, and a new complex (40 kDa) appeared in both control and patient cells cultured in galactose. Re-entry of patient cells into the cell cycle, which occurred after prolonged culture in galactose, was associated with a marked increase in Complex I, and restoration of the oxygen consumption defect. Our results indicate that CHCHD10-CHCHD2 complexes are necessary for efficient mitochondrial respiration, and support a role for mitochondrial dysfunction in some patients with ALS., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mitochondrial protein synthesis is initiated by formylated tRNA-methionine, which requires the activity of MTFMT, a methionyl-tRNA formyltransferase. Mutations in MTFMT have been associated with Leigh syndrome, early-onset mitochondrial leukoencephalopathy, microcephaly, ataxia, and cardiomyopathy. We identified compound heterozygous MTFMT mutations in a patient with a mild neurological phenotype and late-onset progressive visual impairment. MRI studies documented a progressive and selective involvement of the retrochiasmatic visual pathway. MTFMT was undetectable by immunoblot analysis of patient fibroblasts, resulting in specific defects in mitochondrial protein synthesis and assembly of the oxidative phosphorylation complexes. This report expands the clinical and MRI phenotypes associated with MTFMT mutations, illustrating the complexity of genotype-phenotype relationships in mitochondrial translation disorders.

Pseudouridylation is a common post-transcriptional modification in RNA, but its functional consequences at the cellular level remain largely unknown. Using a proximity-biotinylation assay, we identified a protein module in mitochondrial RNA granules, platforms for post-transcriptional RNA modification and ribosome assembly, containing several proteins of unknown function including three uncharacterized pseudouridine synthases, TRUB2, RPUSD3, and RPUSD4. TRUB2 and RPUSD4 were previously identified as core essential genes in CRISPR/Cas9 screens. Depletion of the individual enzymes produced specific mitochondrial protein synthesis and oxidative phosphorylation assembly defects without affecting mitochondrial mRNA levels. Investigation of the molecular targets in mitochondrial RNA by pseudouridine-Seq showed that RPUSD4 plays a role in the pseudouridylation of a single residue in the 16S rRNA, a modification that is essential for its stability and assembly into the mitochondrial ribosome, while TRUB2/RPUSD3 were similarly involved in pseudouridylating specific residues in mitochondrial mRNAs. These results establish essential roles for epitranscriptomic modification of mitochondrial RNA in mitochondrial protein synthesis, oxidative phosphorylation, and cell survival., (© 2016 The Authors.)

Vitamin B12 (cobalamin, Cbl) cofactors adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) are required for the activity of the enzymes methylmalonyl-CoA mutase (MCM) and methionine synthase (MS). Inborn errors of Cbl metabolism are rare Mendelian disorders associated with hematological and neurological manifestations, and elevations of methylmalonic acid and/or homocysteine in the blood and urine. We describe a patient whose fibroblasts had decreased functional activity of MCM and MS and decreased synthesis of AdoCbl and MeCbl (3.4% and 1.0% of cellular Cbl, respectively). The defect in cultured patient fibroblasts complemented those from all known complementation groups. Patient cells accumulated transcobalamin-bound-Cbl, a complex which usually dissociates in the lysosome to release free Cbl. Whole-exome sequencing identified putative disease-causing variants c.851T>G (p.L284*) and c.1019C>T (p.T340I) in transcription factor ZNF143. Proximity biotinylation analysis confirmed the interaction between ZNF143 and HCFC1, a protein that regulates expression of the Cbl trafficking enzyme MMACHC. qRT-PCR analysis revealed low MMACHC expression levels both in patient fibroblasts, and in control fibroblasts incubated with ZNF143 siRNA., (© 2016 WILEY PERIODICALS, INC.)

Mitochondria form a dynamic network that responds to physiological signals and metabolic stresses by altering the balance between fusion and fission. Mitochondrial fusion is orchestrated by conserved GTPases MFN1/2 and OPA1, a process coordinated in yeast by Ugo1, a mitochondrial metabolite carrier family protein. We uncovered a homozygous missense mutation in SLC25A46, the mammalian orthologue of Ugo1, in a subject with Leigh syndrome. SLC25A46 is an integral outer membrane protein that interacts with MFN2, OPA1, and the mitochondrial contact site and cristae organizing system (MICOS) complex. The subject mutation destabilizes the protein, leading to mitochondrial hyperfusion, alterations in endoplasmic reticulum (ER) morphology, impaired cellular respiration, and premature cellular senescence. The MICOS complex is disrupted in subject fibroblasts, resulting in strikingly abnormal mitochondrial architecture, with markedly shortened cristae. SLC25A46 also interacts with the ER membrane protein complex EMC, and phospholipid composition is altered in subject mitochondria. These results show that SLC25A46 plays a role in a mitochondrial/ER pathway that facilitates lipid transfer, and link altered mitochondrial dynamics to early-onset neurodegenerative disease and cell fate decisions., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Complex I (NADH ubiquinone oxidoreductase) is a large multisubunit enzyme that catalyzes the first step in oxidative phosphorylation (OXPHOS). In mammals, complex I biogenesis occurs in a stepwise manner, a process that requires the participation of several nucleus-encoded accessory proteins. The FAD-dependent oxidoreductase-containing domain 1 (FOXRED1) protein is a complex I assembly factor; however, its specific role in the assembly pathway remains poorly understood. We identified a homozygous missense mutation, c.1308 G→A (p.V421M) in FOXRED1 in a patient who presented with epilepsy and severe psychomotor retardation. A patient myoblast line showed a severe reduction in complex I, associated with the accumulation of subassemblies centered around ∼340 kDa, and a milder decrease in complex II, all of which were rescued by retroviral expression of wild-type FOXRED1. Two additional assembly factors, AIFM1 and ACAD9, coimmunoprecipitated with FOXRED1, and all were associated with a 370-kDa complex I subassembly that, together with a 315-kDa subassembly, forms the 550-kDa subcomplex. Loss of FOXRED1 function prevents efficient formation of this midassembly subcomplex. Although we could not identify subassemblies of complex II, our results establish that FOXRED1 function is both broader than expected, involving the assembly of two flavoprotein-containing OXPHOS complexes, and cell type specific., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

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.

Leigh syndrome (LS) is a rare heterogeneous progressive neurodegenerative disorder usually presenting in infancy or early childhood. Clinical presentation is variable and includes psychomotor delay or regression, acute neurological or acidotic episodes, hypotonia, ataxia, spasticity, movement disorders, and corresponding anomalies of the basal ganglia and brain stem on magnetic resonance imaging. To date, 35 genes have been associated with LS, mostly involved in mitochondrial respiratory chain function and encoded in either nuclear or mitochondrial DNA. We used whole-exome sequencing to identify disease-causing variants in four patients with basal ganglia abnormalities and clinical presentations consistent with LS. Compound heterozygote variants in ECHS1, encoding the enzyme enoyl-CoA hydratase were identified. One missense variant (p.Thr180Ala) was common to all four patients and the haplotype surrounding this variant was also shared, suggesting a common ancestor of French-Canadian origin. Rare mutations in ECHS1 as well as in HIBCH, the enzyme downstream in the valine degradation pathway, have been associated with LS or LS-like disorders. A clear clinical overlap is observed between our patients and the reported cases with ECHS1 or HIBCH deficiency. The main clinical features observed in our cohort are T2-hyperintense signal in the globus pallidus and putamen, failure to thrive, developmental delay or regression, and nystagmus. Respiratory chain studies are not strikingly abnormal in our patients: one patient had a mild reduction of complex I and III and another of complex IV. The identification of four additional patients with mutations in ECHS1 highlights the emerging importance of this pathway in LS.

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 Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: We investigated a subject with an isolated cytochrome c oxidase (COX) deficiency presenting with an unusual phenotype characterised by neuropathy, exercise intolerance, obesity, and short stature., Methods and Results: Blue-native polyacrylamide gel electrophoresis (BN-PAGE) analysis showed an almost complete lack of COX assembly in subject fibroblasts, consistent with the very low enzymatic activity, and pulse-labelling mitochondrial translation experiments showed a specific decrease in synthesis of the COX1 subunit, the core catalytic subunit that nucleates assembly of the holoenzyme. Whole exome sequencing identified compound heterozygous mutations (c.199dupC, c.215A>G) in COA3, a small inner membrane COX assembly factor, resulting in a pronounced decrease in the steady-state levels of COA3 protein. Retroviral expression of a wild-type COA3 cDNA completely rescued the COX assembly and mitochondrial translation defects, confirming the pathogenicity of the mutations, and resulted in increased steady-state levels of COX1 in control cells, demonstrating a role for COA3 in the stabilisation of this subunit. COA3 exists in an early COX assembly complex that contains COX1 and other COX assembly factors including COX14 (C12orf62), another single pass transmembrane protein that also plays a role in coupling COX1 synthesis with holoenzyme assembly. Immunoblot analysis showed that COX14 was undetectable in COA3 subject fibroblasts, and that COA3 was undetectable in fibroblasts from a COX14 subject, demonstrating the interdependence of these two COX assembly factors., Conclusions: The mild clinical course in this patient contrasts with nearly all other cases of severe COX assembly defects that are usually fatal early in life, and underscores the marked tissue-specific involvement in mitochondrial diseases., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Cytoplasmic RNA granules play a central role in mRNA metabolism, but the importance of mitochondrial RNA granules remains relatively unexplored. We characterized their proteome and found that they contain a large toolbox of proteins dedicated to RNA metabolism. Investigation of four uncharacterized putative RNA-binding proteins-two RNA helicases, DHX30 and DDX28, and two proteins of the Fas-activated serine-threonine kinase (FASTKD) family, FASTKD2 and FASTKD5-demonstrated that both helicases and FASTKD2 are required for mitochondrial ribosome biogenesis. RNA-sequencing (RNA-seq) analysis showed that DDX28 and FASTKD2 bound the 16S rRNA. FASTKD5 is required for maturing precursor mRNAs that are not flanked by tRNAs and that therefore cannot be processed by the canonical mRNA maturation pathway. Silencing FASTKD5 rendered mature COX I mRNA almost undetectable, which severely reduced the synthesis of COX I, resulting in a complex IV assembly defect. These data demonstrate that mitochondrial RNA granules are centers for posttranscriptional RNA processing and the biogenesis of mitochondrial ribosomes., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS [MIM 270550]) is an early-onset neurodegenerative disorder caused by mutations in the SACS gene. Over 170 SACS mutations have been reported worldwide and are thought to cause loss of function of sacsin, a poorly characterized and massive 520 kDa protein. To establish an animal model and to examine the pathophysiological basis of ARSACS, we generated Sacs knockout (Sacs(-/-)) mice. Null animals displayed an abnormal gait with progressive motor, cerebellar and peripheral nerve dysfunctions highly reminiscent of ARSACS. These clinical features were accompanied by an early onset, progressive loss of cerebellar Purkinje cells followed by spinal motor neuron loss and peripheral neuropathy. Importantly, loss of sacsin function resulted in abnormal accumulation of non-phosphorylated neurofilament (NF) bundles in the somatodendritic regions of vulnerable neuronal populations, a phenotype also observed in an ARSACS brain. Moreover, motor neurons cultured from Sacs(-/-) embryos exhibited a similar NF rearrangement with significant reduction in mitochondrial motility and elongated mitochondria. The data points to alterations in the NF cytoskeleton and defects in mitochondrial dynamics as the underlying pathophysiological basis of ARSACS., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Mitochondrial calcium is an important modulator of cellular metabolism. CCDC90A was reported to be a regulator of the mitochondrial calcium uniporter (MCU) complex, a selective channel that controls mitochondrial calcium uptake, and hence was renamed MCUR1. Here we show that suppression of CCDC90A in human fibroblasts produces a specific cytochrome c oxidase (COX) assembly defect, resulting in decreased mitochondrial membrane potential and reduced mitochondrial calcium uptake capacity. Fibroblasts from patients with COX assembly defects due to mutations in TACO1 or COX10 also showed reduced mitochondrial membrane potential and impaired calcium uptake capacity, both of which were rescued by expression of the respective wild-type cDNAs. Deletion of fmp32, a homolog of CCDC90A in Saccharomyces cerevisiae, an organism that lacks an MCU, also produces a COX deficiency, demonstrating that the function of CCDC90A is evolutionarily conserved. We conclude that CCDC90A plays a role in COX assembly and does not directly regulate MCU., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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., (© 2014 WILEY PERIODICALS, INC.)

Complex I of the mitochondrial respiratory chain is a large multisubunit enzyme that assembles from nuclear and mtDNA-encoded components. Several complex I assembly factors have been identified, but their precise functions are not well understood. Here, we have investigated the function of one of these, NDUFAF7, a soluble matrix protein comprised of a DUF185 domain that harbors a methyltransferase motif. Knockdown of NDUFAF7 by siRNA in human fibroblasts produced a specific complex I assembly defect, as did morpholino-mediated knockdown of the zebrafish ortholog. Germline disruption of the murine ortholog was an early embryonic lethal. The complex I assembly defect was characterized by rapid, AFG3L2-dependent, turnover of newly synthesized ND1, the subunit that seeds the assembly pathway, and by decreased steady-state levels of several other structural subunits including NDUFS2, NDUFS1 and NDUFA9. Expression of an NDUFAF7 mutant (G124V), predicted to disrupt methyltransferase activity, impaired complex I assembly, suggesting an assembly factor or structural subunit as a substrate for methylation. To identify the NDUFAF7 substrate, we used an anti-ND1 antibody to immunoprecipitate complex I and its associated assembly factors, followed by mass spectrometry to detect posttranslational protein modifications. Analysis of an NDUFAF7 methyltransferase mutant showed a 10-fold reduction in an NDUFS2 peptide containing dimethylated Arg85, but a 5-fold reduction in three other NDUFS2 peptides. These results show that NDUFAF7 functions to methylate NDUFS2 after it assembles into a complex I, stabilizing an early intermediate in the assembly pathway, and that this function is essential for normal vertebrate development., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Isolated cytochrome c oxidase (COX) deficiency is a common cause of mitochondrial disease, yet its genetic basis remains unresolved in many patients. Here, we identified novel compound heterozygous mutations in SCO1 (p.M294V, p.Val93*) in one such patient with fatal encephalopathy. The patient lacked the severe hepatopathy (p.P174L) or hypertrophic cardiomyopathy (p.G132S) observed in previously reported SCO1 cases, so we investigated whether allele-specific defects in SCO1 function might underlie the genotype-phenotype relationships. Fibroblasts expressing p.M294V had a relatively modest decrease in COX activity compared with those expressing p.P174L, whereas both SCO1 lines had marked copper deficiencies. Overexpression of known pathogenic variants in SCO1 fibroblasts showed that p.G132S exacerbated the COX deficiency, whereas COX activity was partially or fully restored by p.P174L and p.M294V, respectively. These data suggest that the clinical phenotypes in SCO1 patients might reflect the residual capacity of the pathogenic alleles to perform one or both functions of SCO1., (© 2013 WILEY PERIODICALS, INC.)

RNA-binding proteins are at the heart of posttranscriptional gene regulation, coordinating the processing, storage, and handling of cellular RNAs. We show here that GRSF1, previously implicated in the binding and selective translation of influenza mRNAs, is targeted to mitochondria where it forms granules that colocalize with foci of newly synthesized mtRNA next to mitochondrial nucleoids. GRSF1 preferentially binds RNAs transcribed from three contiguous genes on the light strand of mtDNA, the ND6 mRNA, and the long noncoding RNAs for cytb and ND5, each of which contains multiple consensus binding sequences. RNAi-mediated knockdown of GRSF1 leads to alterations in mitochondrial RNA stability, abnormal loading of mRNAs and lncRNAs on the mitochondrial ribosome, and impaired ribosome assembly. This results in a specific protein synthesis defect and a failure to assemble normal amounts of the oxidative phosphorylation complexes. These data implicate GRSF1 as a key regulator of posttranscriptional mitochondrial gene expression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

SCO1 and SCO2 are metallochaperones whose principal function is to add two copper ions to the catalytic core of cytochrome c oxidase (COX). However, affected tissues of SCO1 and SCO2 patients exhibit a combined deficiency in COX activity and total copper content, suggesting additional roles for these proteins in the regulation of cellular copper homeostasis. Here we show that both the redox state of the copper-binding cysteines of SCO1 and the abundance of SCO2 correlate with cellular copper content and that these relationships are perturbed by mutations in SCO1 or SCO2, producing a state of apparent copper overload. The copper deficiency in SCO patient fibroblasts is rescued by knockdown of ATP7A, a trans-Golgi, copper-transporting ATPase that traffics to the plasma membrane during copper overload to promote efflux. To investigate how a signal from SCO1 could be relayed to ATP7A, we examined the abundance and subcellular distribution of several soluble COX assembly factors. We found that COX19 partitions between mitochondria and the cytosol in a copper-dependent manner and that its knockdown partially rescues the copper deficiency in patient cells. These results demonstrate that COX19 is necessary for the transduction of a SCO1-dependent mitochondrial redox signal that regulates ATP7A-mediated cellular copper efflux.

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.

MMACHC and MMADHC are the genes responsible for cblC and cblD defects of vitamin B(12) metabolism, respectively. Patients with cblC and cblD defects present with various combinations of methylmalonic aciduria (MMA) and homocystinuria (HC). Those with cblC mutations have both MMA and HC whereas cblD patients can present with one of three distinct biochemical phenotypes: isolated MMA, isolated HC, or combined MMA and HC. Based on the subcellular localization of these enzymatic pathways it is thought that MMACHC functions in the cytoplasm while MMADHC functions downstream of MMACHC in both the cytoplasm and the mitochondrion. In this study we determined the subcellular location of MMACHC and MMADHC by immunofluorescence and subcellular fractionation. We show that MMACHC is cytoplasmic while MMADHC is both mitochondrial and cytoplasmic, consistent with the proposal that MMADHC acts as a branch point for vitamin B(12) delivery to the cytoplasm and mitochondria. The factors that determine the distribution of MMADHC between the cytoplasm and mitochondria remain unknown. Functional complementation experiments showed that retroviral expression of the GFP tagged constructs rescued all biochemical defects in cblC and cblD fibroblasts except propionate incorporation in cblD-MMA cells, suggesting that the endogenous mutant protein interferes with the function of the transduced wild type construct., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mitochondrial respiratory-chain complexes assemble from subunits of dual genetic origin assisted by specialized assembly factors. Whereas core subunits are translated on mitochondrial ribosomes, others are imported after cytosolic translation. How imported subunits are ushered to assembly intermediates containing mitochondria-encoded subunits is unresolved. Here, we report a comprehensive dissection of early cytochrome c oxidase assembly intermediates containing proteins required for normal mitochondrial translation and reveal assembly factors promoting biogenesis of human respiratory-chain complexes. We find that TIM21, a subunit of the inner-membrane presequence translocase, is also present in the major assembly intermediates containing newly mitochondria-synthesized and imported respiratory-chain subunits, which we term MITRAC complexes. Human TIM21 is dispensable for protein import but required for integration of early-assembling, presequence-containing subunits into respiratory-chain intermediates. We establish an unexpected molecular link between the TIM23 transport machinery and assembly of respiratory-chain complexes that regulate mitochondrial protein synthesis in response to their assembly state., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Mutations in the genes composing the mitochondrial translation apparatus are an important cause of a heterogeneous group of oxidative phosphorylation (OXPHOS) disorders. We studied the index case in a consanguineous family in which two children presented with severe encephalopathy, lactic acidosis, and intractable seizures leading to an early fatal outcome. Blue native polyacrylamide gel electrophoretic (BN-PAGE) analysis showed assembly defects in all of the OXPHOS complexes with mtDNA-encoded structural subunits, and these defects were associated with a severe deficiency in mitochondrial translation. Immunoblot analysis showed reductions in the steady-state levels of several structural subunits of the mitochondrial ribosome. Whole-exome sequencing identified a homozygous missense mutation (c.1250G>A) in an uncharacterized gene, RMND1 (required for meiotic nuclear division 1). RMND1 localizes to mitochondria and behaves as an integral membrane protein. Retroviral expression of the wild-type RMND1 cDNA rescued the biochemical phenotype in subject cells, and siRNA-mediated knockdown of the protein recapitulated the defect. BN-PAGE, gel filtration, and mass spectrometry analyses showed that RMND1 forms a high-molecular-weight and most likely homopolymeric complex (∼240 kDa) that does not assemble in subject fibroblasts but that is rescued by expression of RMND1 cDNA. The p.Arg417Gln substitution, predicted to be in a coiled-coil domain, which is juxtaposed to a transmembrane domain at the extreme C terminus of the protein, does not alter the steady-state level of RMND1 but might prevent protein-protein interactions in this complex. Our results demonstrate that the RMND1 complex is necessary for mitochondrial translation, possibly by coordinating the assembly or maintenance of the mitochondrial ribosome., (Copyright © 2012 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)