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Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q

Complex I deficiency, the most common respiratory chain defect, is genetically heterogeneous: mutations in 8 nuclear and 7 mitochondrial DNA genes encoding complex I subunits have been described. However, these genes account for disease in only a minority of complex I–deficient patients. We investigated whether there may be an unknown common gene by performing functional complementation analysis of cell lines from 10 unrelated patients. Two of the patients were found to have mitochondrial DNA mutations. The other 8 represented 7 different (nuclear) complementation groups, all but 1 of which showed abnormalities of complex I assembly. It is thus unlikely that any one unknown gene accounts for a large proportion of complex I cases. The 2 patients sharing a nuclear complementation group had a similar abnormal complex I assembly profile and were studied further by homozygosity mapping, chromosome transfers, and microarray expression analysis. NDUFS6, a complex I subunit gene not previously associated with complex I deficiency, was grossly underexpressed in the 2 patient cell lines. Both patients had homozygous mutations in this gene, one causing a splicing abnormality and the other a large deletion. This integrated approach to gene identification offers promise for identifying other unknown causes of respiratory chain disorders.

The mitochondrial intermembrane space protein AIFM1 has been reported to mediate the import of MIA40/CHCHD4, which forms the import receptor in the mitochondrial disulfide relay. Here, we demonstrate that AIFM1 and MIA40/CHCHD4 cooperate beyond this MIA40/CHCHD4 import. We show that AIFM1 and MIA40/CHCHD4 form a stable long-lived complex in vitro, in different cell lines, and in tissues. In HEK293 cells lacking AIFM1, levels of MIA40 are unchanged, but the protein is present in the monomeric form. Monomeric MIA40 neither efficiently interacts with nor mediates the import of specific substrates. The import defect is especially severe for NDUFS5, a subunit of complex I of the respiratory chain. As a consequence, NDUFS5 accumulates in the cytosol and undergoes rapid proteasomal degradation. Lack of mitochondrial NDUFS5 in turn results in stalling of complex I assembly. Collectively, we demonstrate that AIFM1 serves two overlapping functions: importing MIA40/CHCHD4 and constituting an integral part of the disulfide relay that ensures efficient interaction of MIA40/CHCHD4 with specific substrates.

Mitochondria form dynamic networks in the cell that are balanced by the flux of iterative fusion and fission events of the organelles. It is now appreciated that mitochondrial fission also represents an end-point event in a signalling axis that allows cells to sense and respond to external cues. The fission process is orchestrated by membrane-associated adaptors, influenced by organellar and cytoskeletal interactions and ultimately executed by the dynamin-like GTPase DRP1. Here we invoke the framework of the 'mitochondrial divisome', which is conceptually and operationally similar to the bacterial cell-division machinery. We review the functional and regulatory aspects of the mitochondrial divisome and, within this framework, parse the core from the accessory machinery. In so doing, we transition from a phenomenological to a mechanistic understanding of the fission process.

PURPOSE The modern researcher is confronted with hundreds of published methods to interpret genetic variants. There are databases of genes and variants, phenotype-genotype relationships, algorithms that score and rank genes, and in silico variant effect prediction tools. Because variant prioritization is a multifactorial problem, a welcome development in the field has been the emergence of decision support frameworks, which make it easier to integrate multiple resources in an interactive environment. Current decision support frameworks are typically limited by closed proprietary architectures, access to a restricted set of tools, lack of customizability, Web dependencies that expose protected data, or limited scalability. METHODS We present the Open Custom Ranked Analysis of Variants Toolkit 1 (OpenCRAVAT) a new open-source, scalable decision support system for variant and gene prioritization. We have designed the resource catalog to be open and modular to maximize community and developer involvement, and as a result, the catalog is being actively developed and growing every month. Resources made available via the store are well suited for analysis of cancer, as well as Mendelian and complex diseases. RESULTS OpenCRAVAT offers both command-line utility and dynamic graphical user interface, allowing users to install with a single command, easily download tools from an extensive resource catalog, create customized pipelines, and explore results in a richly detailed viewing environment. We present several case studies to illustrate the design of custom workflows to prioritize genes and variants. CONCLUSION OpenCRAVAT is distinguished from similar tools by its capabilities to access and integrate an unprecedented amount of diverse data resources and computational prediction methods, which span germline, somatic, common, rare, coding, and noncoding variants.

Significance Mitochondria are double-membraned eukaryotic organelles that house the proteins required for generation of ATP, the energy currency of cells. ATP generation within mitochondria is performed by five multisubunit complexes (complexes I to V), the assembly of which is an intricate process. Mutations in subunits of these complexes, or the suite of proteins that help them assemble, lead to a severe multisystem condition called mitochondrial disease. We show that SFXN4, a protein that causes mitochondrial disease when mutated, assists with the assembly of complex I. This finding explains why mutations in SFXN4 cause mitochondrial disease and is surprising because SFXN4 belongs to a family of amino acid transporter proteins, suggesting that it has undergone a dramatic shift in function through evolution.

Mitochondrial complex I deficiency is caused by pathogenic variants in mitochondrial and nuclear genes associated with complex I structure and assembly. We report the case of a patient with NDUFA8-related mitochondrial disease. The patient presented with developmental delay, microcephaly, and epilepsy. His fibroblasts showed apparent biochemical defects in mitochondrial complex I. Whole-exome sequencing revealed that the patient carried a homozygous variant in NDUFA8. His fibroblasts showed a reduction in the protein expression level of not only NDUFA8, but also the other complex I subunits, consistent with assembly defects. The enzyme activity of complex I and oxygen consumption rate were restored by reintroducing wild-typeNDUFA8 cDNA into patient fibroblasts. The functional properties of the variant in NDUFA8 were also investigated using NDUFA8 knockout cells expressing wild-type or mutated NDUFA8 cDNA. These experiments further supported the pathogenicity of the variant in complex I assembly. This is the first report describing that the loss of NDUFA8, which has not previously been associated with mitochondrial disease, causes severe defect in the assembly of mitochondrial complex I, leading to progressive neurological and developmental abnormalities.

NDUFAB1 is the mitochondrial acyl carrier protein (ACP) essential for cell viability. Through its pantetheine-4′-phosphate post-translational modification, NDUFAB1 interacts with members of the leucine-tyrosine-arginine motif (LYRM) protein family. Although several LYRM proteins have been described to participate in a variety of defined processes, the functions of others remain either partially or entirely unknown. We profiled the interaction network of NDUFAB1 to reveal associations with 9 known LYRM proteins as well as more than 20 other proteins involved in mitochondrial respiratory chain complex and mitochondrial ribosome assembly. Subsequent knockout and interaction network studies in human cells revealed the LYRM member AltMiD51 to be important for optimal assembly of the large mitoribosome subunit, consistent with recent structural studies. In addition, we used proteomics coupled with topographical heat-mapping to reveal that knockout of LYRM2 impairs assembly of the NADH-dehydrogenase module of complex I, leading to defects in cellular respiration. Together, this work adds to the catalogue of functions executed by LYRM family of proteins in building mitochondrial complexes and emphasizes the common and essential role of NDUFAB1 as a protagonist in mitochondrial metabolism.

Caveolae have been linked to many biological functions, but their precise roles are unclear. Using quantitative whole cell proteomics of genome-edited cells, we show that the oxidative stress response is the major pathway dysregulated in cells lacking the key caveola structural protein, CAVIN1. CAVIN1 deletion compromised sensitivity to oxidative stress in cultured cells and in animals. Wound-induced accumulation of reactive oxygen species and apoptosis were suppressed in Cavin1-null zebrafish, negatively affecting regeneration. Oxidative stress triggered lipid peroxidation and induced caveolar disassembly. The resulting release of CAVIN1 from caveolae allowed direct interaction between CAVIN1 and NRF2, a key regulator of the antioxidant response, facilitating NRF2 degradation. CAVIN1-null cells with impaired negative regulation of NRF2 showed resistance to lipid peroxidation-induced ferroptosis. Thus, caveolae, via lipid peroxidation and CAVIN1 release, maintain cellular susceptibility to oxidative stress-induced cell death demonstrating a crucial role for this enigmatic organelle in cellular homeostasis and wound response.

Cytochrome c oxidase assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked in patients to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here we show that the absence of COA7 leads to arrest of the complex IV assembly pathway at the initial step where the COX1 module is built, which requires incorporation of copper and heme cofactors. In solution, purified COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. Surprisingly, the crystal structure of COA7, determined to 2.4 Å resolution, reveals a ‘banana-shaped’ molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds, with a structure entirely distinct from proteins with characterized heme binding activities. We therefore propose a role for COA7 in heme binding/chaperoning in the mitochondrial intermembrane space, this activity being crucial for and providing a missing link in complex IV biogenesis.Significance StatementAssembly factors play key roles in the biogenesis of many mitochondrial protein complexes regulating their stability, activity and incorporation of essential cofactors. COA7 is a metazoan-specific assembly factor, the absence or mutation of which in humans accompanies complex IV assembly defects and neurological conditions. Here we report the crystal structure of COA7 to 2.4 Å resolution, revealing a ‘banana-shaped’ molecule composed of five helix-turn-helix (α/α) repeats, tethered by disulfide bonds. Characterization of pathogenic variants reveals significantly lower stabilities, correlating with the associated disease outcomes. Fascinatingly, COA7 binds heme with micromolar affinity, despite the fact that the protein structure does not resemble previously characterized heme-binding proteins. This provides a possible missing link for heme handling in the mitochondrial intermembrane space.

Caveolae-associated protein 3 (cavin3) is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair, with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays revealed a direct interaction between BRCA1 and cavin3 that occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. Supporting a role in DNA repair, cavin3-deficient cells were sensitive to PARP inhibition, where concomitant depletion of 53BP1 restored BRCA1-dependent sensitivity to PARP inhibition. We conclude that cavin3 functions together with BRCA1 in multiple cancer-related pathways. The loss of cavin3 function may provide tumor cell survival by attenuating apoptotic sensitivity and hindering DNA repair under chronic stress conditions., eLife digest When cells become cancerous they often stop making certain proteins. This includes a protein known as cavin3 which resides in bulb-shaped pits of the membrane that surrounds the cell called caveolae. These structures work like stress detectors, picking up changes in the membrane and releasing proteins, such as cavin3, into the cell’s interior. Past studies suggest that cavin3 might interact with a protein called BRCA1 that suppresses the formation of tumors. Cells with mutations in the gene for BRCA1 struggle to fix damage in their DNA, and have to rely on other repair proteins, such as PARPs (short for poly (ADP-ribose) polymerases). Blocking PARP proteins with drugs can kill cancer cells with problems in their BRCA1 proteins. However, it was unclear what role cavin3 plays in this mechanism. To investigate this, McMahon et al. exposed cells grown in the laboratory to DNA-damaging UV light to stimulate the release of cavin3 from caveolae. This revealed that cavin3 interacts with BRCA1 when cells are under stress, and helps stabilize the protein so it can perform DNA repairs. Cells without cavin3 showed decreased levels of the BRCA1 protein, but compensated for the loss of BRCA1 by increasing the levels of their PARP proteins. These cells also had increased DNA damage following treatment with drugs that block PARPs, similar to cancer cells carrying mutations in the gene for BRCA1. These findings suggest that cavin3 helps BRCA1 to suppress the formation of tumors, and therefore should be considered when developing new anti-cancer treatments.

Mitochondrial disease is a debilitating condition with a diverse genetic aetiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labelling interaction studies reveal that TMEM126A associates with the newly synthesised mtDNA-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. Importantly, we clarify that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I, helping to explain the differences in disease aetiology observed between these two genes.

The structural and functional organization of the mitochondrial respiratory chain (MRC) remains intensely debated. Here, we show the co-existence of two separate MRC organizations in human cells and postmitotic tissues, C-MRC and S-MRC, defined by the preferential expression of three COX7A subunit isoforms, COX7A1/2 and SCAFI (COX7A2L). COX7A isoforms promote the functional reorganization of distinct co-existing MRC structures to prevent metabolic exhaustion and MRC deficiency. Notably, prevalence of each MRC organization is reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC). Under oxidative conditions, the C-MRC is bioenergetically more efficient, whereas the S-MRC preferentially maintains oxidative phosphorylation (OXPHOS) upon metabolic rewiring toward glycolysis. We show a link between the metabolic signatures converging at the PDC and the structural and functional organization of the MRC, challenging the widespread notion of the MRC as a single functional unit and concluding that its structural heterogeneity warrants optimal adaptation to metabolic function.

In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. TheWhole exome, whole genome and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease.We report six differentAustralian NHMRC, US Department of Defense, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance and Australian Mito Foundation.

Summary Background In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The ATAD3 locus is one such region in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively. Methods Whole-exome, whole-genome, and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease. Findings We report 6 different de novo duplications in the ATAD3 gene locus causing a distinctive presentation, including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently, corneal clouding or cataracts and encephalopathy. The recurrent 68-kb ATAD3 duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The ATAD3 duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes, and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue. Conclusions ATAD3 duplications appear to act in a dominant-negative manner and the de novo inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience, the ATAD3 locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease, but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies. Funding Australian NHMRC, US Department of Defense, US National Institutes of Health, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance, and Australian Mito Foundation.

Virtual memory T (TVM) cells are antigen-naïve CD8+ T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (TMEM) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of TVM cells and their altered functionality with age, here we investigate TVM cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of TVM, but not TMEM, cells and it increases with age in both subsets. The elevated SRC observed in aged mouse TVM cells and human CD8+ T cells from older individuals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of TVM, but not TMEM, cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8+ T cells., Fatty acid oxidation (FAO) is thought to contribute to high spare respiratory capacity (SRC), which in turn affects CD8+ T cell function. Here, the authors show that ex vivo virtual memory T cells (and not antigen experienced memory T cells) have high SRC, a metabolic state that it is affected by ageing and IL-15 signalling and not directly by FAO.

Complex IV (cytochrome c oxidase; COX) is the terminal complex of the mitochondrial electron transport chain. Copper is essential for COX assembly, activity, and stability, and is incorporated into the dinuclear CuA and mononuclear CuB sites. Multiple assembly factors play roles in the biogenesis of these sites within COX and the failure of this intricate process, such as through mutations to these factors, disrupts COX assembly and activity. Various studies over the last ten years have revealed that the assembly factor COA6, a small intermembrane space-located protein with a twin CX9C motif, plays a role in the biogenesis of the CuA site. However, how COA6 and its copper binding properties contribute to the assembly of this site has been a controversial area of research. In this review, we summarize our current understanding of the molecular mechanisms by which COA6 participates in COX biogenesis.

Caveolae-associated protein 3 (cavin3), a putative tumor suppressor protein, is inactivated in most cancers. We characterized how cavin3 affects the cellular proteome using genome-edited cells together with label-free quantitative proteomics. These studies revealed a prominent role for cavin3 in DNA repair with BRCA1 and BRCA1 A-complex components being downregulated on cavin3 deletion. Cellular and cell-free expression assays, we show a direct interaction between BRCA1 and cavin3. Association of BRCA1 and cavin3 occurs when cavin3 is released from caveolae that are disassembled in response to UV and mechanical stress. Supporting a role in DNA repair, cavin3-deficient cells were sensitized to the effects of PARP inhibition, which compromises DNA repair, and showed reduced recruitment of the BRCA1 A-complex to UV DNA damage foci. Overexpression and RNAi-depletion revealed that cavin3 sensitized various cancer cells to UV-induced apoptosis. We conclude that cavin3 functions together with BRCA1 in multiple pathways that contribute to tumorigenesis.

The many functions of mitochondria—the powerhouses of our cells—are intimately linked with their ultrastructure and network morphology. In this issue, Stephan et al (2020) apply a tour de force of microscopic techniques to examine the contributions of specific mitochondrial proteins to crista architecture.

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. Several assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. Although in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

The ‘mitochondrial contact site and cristae organising system’ (MICOS) is an essential protein complex that promotes the formation, maintenance and stability of mitochondrial cristae. As such, loss of core MICOS components disrupts cristae structure and impairs mitochondrial function. Aberrant mitochondrial cristae morphology and diminished mitochondrial function is a pathological hallmark observed across many human diseases such as neurodegenerative conditions, obesity and diabetes mellitus, cardiomyopathy, and in muscular dystrophies and myopathies. While mitochondrial abnormalities are often an associated secondary effect to the pathological disease process, a direct role for the MICOS in health and human disease is emerging. This review describes the role of MICOS in the maintenance of mitochondrial architecture and summarizes both the direct and associated roles of the MICOS in human disease.

Human Tim8a and Tim8b are members of an intermembrane space chaperone network, known as the small TIM family. Mutations in TIMM8A cause a neurodegenerative disease, Mohr-Tranebjærg syndrome (MTS), which is characterised by sensorineural hearing loss, dystonia and blindness. Nothing is known about the function of hTim8a in neuronal cells or how mutation of this protein leads to a neurodegenerative disease. We show that hTim8a is required for the assembly of Complex IV in neurons, which is mediated through a transient interaction with Complex IV assembly factors, in particular the copper chaperone COX17. Complex IV assembly defects resulting from loss of hTim8a leads to oxidative stress and changes to key apoptotic regulators, including cytochrome c, which primes cells for death. Alleviation of oxidative stress with Vitamin E treatment rescues cells from apoptotic vulnerability. We hypothesise that enhanced sensitivity of neuronal cells to apoptosis is the underlying mechanism of MTS.

Summary: Mitochondrial complex I harbors 7 mitochondrial and 38 nuclear-encoded subunits. Its biogenesis requires the assembly and integration of distinct intermediate modules, mediated by numerous assembly factors. The mitochondrial complex I intermediate assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module. The role of the MCIA complex and the involvement of other proteins in the biogenesis of this module is unclear. Cell knockout studies reveal that while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centered on ACAD9. We also identify TMEM186 and COA1 as bona fide components of the MCIA complex with loss of either resulting in MCIA complex defects and reduced complex I assembly. TMEM186 enriches with newly translated ND3, and COA1 enriches with ND2. Our findings provide new functional insights into the essential nature of the MCIA complex in complex I assembly. : Formosa et al. investigate the function of the MCIA complex in complex I assembly. They demonstrate the requirement of individual components for the formation of complex I intermediates and assembly of the final enzyme. Finally, they characterize the involvement of TMEM186 and COA1 in this process. Keywords: assembly factors, complex I, MCIA complex, mitochondria, NADH-ubiquinone dehydrogenase, oxidative phosphorylation

PURPOSEThe modern researcher is confronted with hundreds of published methods to interpret genetic variants. There are databases of genes and variants, phenotype-genotype relationships, algorithms that score and rank genes, and in silico variant effect prediction tools. Because variant prioritization is a multi-factorial problem, a welcome development in the field has been the emergence of decision support frameworks, which make it easier to integrate multiple resources in an interactive environment. Current decision support frameworks are typically limited by closed proprietary architectures, access to a restricted set of tools, lack of customizability, web dependencies that expose protected data, or limited scalability.METHODSWe present OpenCRAVAT, a new open source, scalable decision support system for variant and gene prioritization. We have designed the resource catalog to be open and modular to maximize community and developer involvement, and as a result the catalog is being actively developed and growing every month. Resources made available via the store are well-suited for analysis of cancer, as well as Mendelian and complex diseases.RESULTSOpenCRAVAT offers both command line utility and dynamic GUI, allowing users to install with a single command, easily download tools from an extensive resource catalog, create customized pipelines, and explore results in a richly detailed viewing environment. We present several case studies to illustrate the design of custom workflows to prioritize genes and variants.CONCLUSIONOpenCRAVAT is distinguished from similar tools by its capabilities to access and integrate an unprecedented amount of diverse data resources and computational prediction methods, which span germline, somatic, common, rare, coding and non-coding variants. OpenCRAVAT is freely available at https://opencravat.org

Mitochondrial complex I is the primary entry point for electrons into the electron transport chain, required for the bulk of cellular ATP production via oxidative phosphorylation. Complex I consists of 45 subunits, which are encoded by both nuclear and mitochondrial DNA. Currently, at least 15 assembly factors are known to be required for the complete maturation of complex I. Mutations in the genes encoding subunits and assembly factors lead to complex I deficiency, which can manifest as mitochondrial disease. The current model of complex I assembly suggests that the enzyme is built by the association of a set of smaller intermediate modules containing specific conserved core subunits and additional accessory subunits. Each module must converge in a spatially and temporally orchestrated fashion to allow assembly of the mature holoenzyme to occur. This review outlines the current understanding of complex I biogenesis, with an emphasis on the assembly factors that facilitate the building of this architectural giant.

Human induced pluripotent stem cells (iPSCs) are a valuable tool for studying the cardiac developmental process in vitro, and cardiomyocytes derived from iPSCs are a putative cell source for personalized medicine. Changes in mitochondrial morphology have been shown to occur during cellular reprogramming and pluripotent stem cell differentiation. However, the relationships between mitochondrial dynamics and cardiac mesoderm commitment of iPSCs remain unclear. Here we demonstrate that changes in mitochondrial morphology from a small granular fragmented phenotype in pluripotent stem cells to a filamentous reticular elongated network in differentiated cardiomyocytes are required for cardiac mesodermal differentiation. Genetic and pharmacological inhibition of the mitochondrial fission protein, Drp1, by either small interfering RNA or Mdivi-1, respectively, increased cardiac mesoderm gene expression in iPSCs. Treatment of iPSCs with Mdivi-1 during embryoid body formation significantly increased the percentage of beating embryoid bodies and expression of cardiac-specific genes. Furthermore, Drp1 gene silencing was accompanied by increased mitochondrial respiration and decreased aerobic glycolysis. Our findings demonstrate that shifting the balance of mitochondrial morphology toward fusion by inhibition of Drp1 promoted cardiac differentiation of human iPSCs with a metabolic shift from glycolysis towards oxidative phosphorylation. These findings suggest that Drp1 may represent a new molecular target for future development of strategies to promote the differentiation of human iPSCs into cardiac lineages for patient-specific cardiac regenerative medicine.

Summary It is increasingly appreciated that alternative splicing plays a key role in generating functional specificity and diversity in cancer. However, the mechanisms by which cancer mutations perturb splicing remain unknown. Here, we developed a network-based strategy, DrAS-Net, to investigate more than 2.5 million variants across cancer types and link somatic mutations with cancer-specific splicing events. We identified more than 40,000 driver variant candidates and their 80,000 putative splicing targets deregulated in 33 cancer types and inferred their functional impact. Strikingly, tumors with splicing perturbations show reduced expression of immune system-related genes and increased expression of cell proliferation markers. Tumors harboring different mutations in the same gene often exhibit distinct splicing perturbations. Further stratification of 10,000 patients based on their mutation-splicing relationships identifies subtypes with distinct clinical features, including survival rates. Our work reveals how single-nucleotide changes can alter the repertoires of splicing isoforms, providing insights into oncogenic mechanisms for precision medicine.

The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III2 + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFIKO) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III2 + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFIKO cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells.