Large-scale mitochondrial DNA (mtDNA) deletions can result in deficiency of oxidative phosphorylation and subsequent mitochondrial dysfunction, ultimately leading to mitochondrial disease. To investigate effective treatments, we report a characterised heteroplasmic iPSC-derived neuronal model with a single, large scale ∼6 kb mtDNA deletion. While mtDNA heteroplasmy remains stable during iNGN2-induced neuronal differentiation from iPSCs, the presence of this mtDNA deletion results in an upregulation of mtDNA copy number and compensatory adaptation of oxidative phosphorylation (OXPHOS) machinery. Despite this increase, mitochondrial dysfunction and reduced oxygen consumption is prevalent. Furthermore, as differentiated neurons mature over time, mitochondrial supercomplexes and isolated complex II diminish, suggesting an increase of severity of the mitochondrial dysfunction. In summary, this study provides insight into a novel compensatory mechanism during iPSC differentiation to bypass mitochondrial dysfunction, and how this response exacerbates dysfunction during culture of mature neurons.

A biochemical deficiency of mitochondrial complex I (CI) underlies approximately 30% of cases of primary mitochondrial disease, yet the inventory of molecular machinery required for CI assembly remains incomplete. We previously characterised patients with isolated CI deficiency caused by segregating variants in RTN4IP1, a gene that encodes a mitochondrial NAD(P)H oxidoreductase. Here, we demonstrate that RTN4IP1 deficiency causes a CI assembly defect in both patient fibroblasts and knockout cells, and report that RTN4IP1 is a bona fide CI assembly factor. Complexome profiling revealed accumulation of unincorporated ND5-module and impaired N-module production. RTN4IP1 patient fibroblasts also exhibited defective coenzyme Q biosynthesis, substantiating a second function of RTN4IP1. Thus, our data reveal RTN4IP1 plays necessary and independent roles in both the terminal stages of CI assembly and in coenzyme Q metabolism, and that pathogenic RTN4IP1 variants impair both functions in patients with mitochondrial disease.

A biochemical deficiency of mitochondrial complex I (CI) underlies ~30% of cases of primary mitochondrial disease, yet the inventory of molecular machinery required for CI assembly remains incomplete. We previously characterised patients with isolated CI deficiency caused by segregating variants in RTN4IP1, encoding a mitochondrial NAD(P)H oxidoreductase. Here, we demonstrate that RTN4IP1 deficiency causes a CI assembly defect in both patient fibroblasts and knockout cells, and report that RTN4IP1 is a bona fide CI assembly factor. Complexome profiling revealed accumulation of unincorporated ND5-module and impaired N-module production. RTN4IP1 patient fibroblasts also exhibited defective coenzyme Q biosynthesis, substantiating this emerging function of RTN4IP1. Thus, our data reveal RTN4IP1 plays an essential role in both the terminal stages of CI assembly and in coenzyme Q metabolism, and that pathogenic RTN4IP1 variants impair both functions in patients with mitochondrial disease. ### Competing Interest Statement The authors have declared no competing interest.

Induced pluripotent stem cells (iPSCs) are of significant value due to their wide ranging potential, removing the need for embryonic material. To successfully culture, expand and differentiate these cells, it is crucial to maintain a precise biological environment, including an appropriate attachment substrate. Commonly used attachment substrates include recombinant extracellular matrix (ECM) components like vitronectin, as well as animal-derived ECM mixes such as GelTrex and Matrigel. However, there is growing interest in exploring alternative approaches to support bioactivity of cells. One approach that is gaining traction is the use of the Caf1 protein of Yersinia pestis . This protein is appealing primarily due to its stability, modularity, and ease of production. In this study, we have developed novel variants of Caf1 that effectively support the growth and differentiation of iPSCs, performing at least as well as GelTrex. Our findings highlight the potential of Caf1 laminin and vitronectin mimics as viable alternatives for supporting iPSC growth and differentiation. The successful development of these Caf1 variants opens new avenues for the field, paving the way for better defined, more cost-effective and readily available attachment substrates in iPSC research and applications. ### Competing Interest Statement DTP, HW and JHL are directors of MarraBio Ltd., a spinout company set up to commercialise the Caf1 technology. The company had no role in the funding or design of the study.

The vast majority of oxygen-utilizing eukaryotes need to express their own mitochondrial genome, mtDNA, to survive. In comparison to size of their nuclear genome, mtDNA is minimal, even in the most exceptional examples. Having evolved from bacteria in an endosymbiotic event, it might be expected that the process of mtDNA expression would be relatively simple. The aim of this short review is to illustrate just how wrong this assumption is. The production of functional mitochondrial RNA across species evolved in many directions. Organelles use a dizzying array of RNA processing, modifying, editing, splicing and maturation events that largely require the import of nuclear-encoded proteins from the cytosol. These processes are sometimes driven by the unusual behaviour of the mitochondrial genome from which the RNA is originally transcribed, but in many examples the complex processes that are essential for the production of functional RNA in the organelle, are fascinating and bewildering.

High-resolution imaging has enabled scientists to explore the mitochondrial network at remarkable resolution. This has been exploited to help increase our knowledge of how mitochondrial gene expression is compartmentalized in cultured cells. Here, we provide detailed methodology to simultaneously visualize up to four components including mtDNA-encoded transcripts, submitochondrial marker proteins, mitoribosomal subunits, or core members of the translational apparatus using STED super-resolution nanoscopy.

Mitochondrial protein synthesis is essential for the life of aerobic eukaryotes. Without it, oxidative phosphorylation cannot be coupled. Evolution has shaped a battery of factors and machinery that are key to production of just a handful of critical proteins. In this general concept chapter, we attempt to briefly summarize our current knowledge of the overall process in mitochondria from a variety of species, breaking this down to the four parts of translation: initiation, elongation, termination, and recycling. Where appropriate, we highlight differences between species and emphasize gaps in our understanding. Excitingly, with the current revolution in cryoelectron microscopy and mitochondrial genome editing, it is highly likely that many of these gaps will be resolved in the near future. However, the absence of a faithful in vitro reconstituted system to study mitochondrial translation is still problematic.

Human mitochondria are highly dynamic organelles, fusing and budding to maintain reticular networks throughout many cell types. Although extending to the extremities of the cell, the majority of the network is concentrated around the nucleus in most of the commonly cultured cell lines. This organelle harbours its own genome, mtDNA, with a different gene content to the nucleus, but the expression of which is critical for maintaining oxidative phosphorylation. Recent advances in click chemistry have allowed us to visualise sites of mitochondrial protein synthesis in intact cultured cells. We show that the majority of translation occurs in the peri-nuclear region of the network. Further analysis reveals that whilst there is a slight peri-nuclear enrichment in the levels of mitoribosomal protein and mitochondrial rRNA, it is not sufficient to explain this substantial heterogeneity in the distribution of translation. Finally, we also show that in contrast, a mitochondrial mRNA does not show such a distinct gradient in distribution. These data suggest that the relative lack of translation in the peripheral mitochondrial network is not due to an absence of mitoribosomes or an insufficient supply of the mt-mRNA transcripts.

Human mitochondria contain their own genome, mitochondrial DNA, that is expressed in the mitochondrial matrix. This genome encodes 13 vital polypeptides that are components of the multi-subunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially encoded components are also integral members of these complexes, where does protein synthesis occur? As transcription, mRNA processing, maturation, and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites? We have adapted a click chemistry-based method coupled with stimulated emission depletion nanoscopy to address these questions. We report that, in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis is detected at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.

Mitochondrial diseases are clinically heterogeneous disorders caused by a wide spectrum of mutations in genes encoded by either the nuclear or the mitochondrial genome. Treatments for mitochondrial diseases are currently focused on symptomatic management rather than improving the biochemical defect caused by a particular mutation. This review focuses on the latest advances in the development of treatments for mitochondrial disease, both small molecules and gene therapies, as well as methods to prevent transmission of mitochondrial disease through the germline.

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?

In mammalian mitochondria, messenger RNA is processed and matured from large primary transcripts in structures known as RNA granules. The identity of the factors and process transferring the matured mRNA to the mitoribosome for translation is unclear. Nascent mature transcripts are believed to associate initially with the small mitoribosomal subunit prior to recruitment of the large subunit to form the translationally active monosome. When the small subunit fails to assemble, however, the stability of mt-mRNA is only marginally affected, and under these conditions, the LRPPRC/SLIRP RNA-binding complex has been implicated in maintaining mt-mRNA stability. Here, we exploit the activity of a bacterial ribotoxin, VapC20, to show that in the absence of the large mitoribosomal subunit, mt-mRNA species are selectively lost. Further, if the small subunit is also depleted, the mt-mRNA levels are recovered. As a consequence of these data, we suggest a natural pathway for loading processed mt-mRNA onto the mitoribosome.

Transplantation of functional mitochondria directly into defective cells is a novel approach that has recently caught the attention of scientists and the general public alike. Could this be too good to be true?

Mitochondrial diseases are progressive, often devastating disorders for which there are currently no cures. The pathogenic mutations responsible can arise in either the nuclear or the mitochondrial genome. For sequence changes in nuclear genes, a molecular therapy mediated by the delivery of a correct copy or by approaches that cause enhanced expression of a different gene that has compensatory effects represent possible therapeutic interventions. In this issue of Molecular Therapy, the article by Luna-Sanchez etal.1Luna-Sanchez M. Benincá C. Cerutti R. Brea-Calvo G. Yeates A. Scorrano L. Zeviani M. Viscomi C. Opa1 Overexpression Protects from Early-Onset Mpv17-/--Related Mouse Kidney Disease.Mol. Ther. 2020; 28 (this issue): 1918-1930Abstract Full Text Full Text PDF Scopus (4) Google Scholar evaluates the potential efficacy of treating a rare disorder caused by mutations in the nuclear-encoded MPV17 gene. MPV17 is a protein found in the inner of the two mitochondrial membranes, but its functions remain obscure. Disease caused by dysfunctional MPV17 has thus far only been reported in 100 patients and is found at the highest density in a restricted region of the southwestern US.2Umetsu S. Inui A. Kobayashi S. Shimura M. Uehara T. Uchida H. Irie R. Sogo T. Komatsu H. Yoshioka T. et al.First cases of MPV17 related mitochondrial DNA depletion syndrome with compound heterozygous mutations in p.R50Q/p.R50W: a case report.Hepatoma Res. 2020; (Published January 6, 2020)https://doi.org/10.20517/2394-5079.2019.030Crossref Google Scholar,3El-Hattab A.W. Wang J. Dai H. Almannai M. Staufner C. Alfadhel M. Gambello M.J. Prasun P. Raza S. Lyons H.J. et al.MPV17-related mitochondrial DNA maintenance defect: New cases and review of clinical, biochemical, and molecular aspects.Hum. Mutat. 2018; 39: 461-470Crossref PubMed Scopus (30) Google Scholar Survival beyond infancy or early childhood is rare and, although symptoms manifest as a broad spectrum of defects, a relatively common feature is the instability of the mitochondrial genome (mtDNA). This is particularly acute in the liver, which Luna-Sanchez etal.1Luna-Sanchez M. Benincá C. Cerutti R. Brea-Calvo G. Yeates A. Scorrano L. Zeviani M. Viscomi C. Opa1 Overexpression Protects from Early-Onset Mpv17-/--Related Mouse Kidney Disease.Mol. Ther. 2020; 28 (this issue): 1918-1930Abstract Full Text Full Text PDF Scopus (4) Google Scholar suggest is due to aberrant infolding of the inner mitochondrial membrane (IMM). They report that, in a mouse lacking Mpv17, the mtDNA levels were partially rescued, and the average life expectancy of the mouse was substantially increased following moderate overexpression of Opa1, a protein responsible for sculpting the IMM. The authors generated an Mpv17 knockout (KO) mouse model (Mpv17−/−) that recapitulated the mtDNA depletion phenotype, albeit strongly in kidney as well as liver. Through various crosses, a further mouse model was generated that still lacked Mpv17 but also overexpressed Opa1, another IMM protein. Opa1 was selected as a potential suppressor of the Mpv17 defect as it is known to play a crucial role in the architecture of the IMM to which mtDNA is anchored.4Gerhold J.M. Cansiz-Arda Ş. Lõhmus M. Engberg O. Reyes A. van Rennes H. Sanz A. Holt I.J. Cooper H.M. Spelbrink J.N. Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure.Sci. Rep. 2015; 5: 15292Crossref PubMed Scopus (65) Google Scholar Previous results from this same group had revealed that overexpression of Opa1 in mouse models with mitochondrial respiratory chain defects had shown mild improvement, which they proposed resulted from an improved environment for the defective multisubunit respiratory chain complex that is normally embedded in the IMM.5Civiletto G. Varanita T. Cerutti R. Gorletta T. Barbaro S. Marchet S. Lamperti C. Viscomi C. Scorrano L. Zeviani M. Opa1 overexpression ameliorates the phenotype of two mitochondrial disease mouse models.Cell Metab. 2015; 21: 845-854Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar The current study was based on a similar premise. Their hypothesis was that a disturbance of the shape and number of infoldings of the IMM, termed cristae (see Figure 1), may potentially affect the mitochondrial genome’s stability. Consequently, restoring the natural structure of the IMM by moderate Opa1 overexpression may lead to an improvement in efficiency of mtDNA replication and transcription (discussed in Nicholls and Gustafsson6Nicholls T.J. Gustafsson C.M. Separating and Segregating the Human Mitochondrial Genome.Trends Biochem. Sci. 2018; 43: 869-881Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Data indicated that overexpression caused (1) an increased cristae volume within the mitochondrion; (2) a narrowing of the cristae junction where it joins the inner boundary membrane of the IMM (see Figure 1), thus potentially restoring the biophysical separation of the cristae compartment from the intermembrane space; and (3) a subsequent elevation of the mtDNA copy number that, in some tissues, approached control levels. Most positive effects were found in kidney tissues where the mtDNA depletion and other physiological defects in the Mpv1−/− mice were pronounced, unlike the situation in MPV1 patients, where kidney defects are uncommon. Is it likely that a moderate overexpression of Opa1 will, therefore, be of therapeutic benefit to patients with disorders resulting from MPV1 mutations? What might be the caveats to this possibility? The mouse model recapitulates the mtDNA depletion, but the predominantly affected tissue is kidney. This contrasts with the human condition, where the vast majority of the patients display hepato-cerebral involvement, where liver problems are the most severe and are consistent with the major depletion of mtDNA being in the liver. Although Opa1 expression in Mpv17 KO background doubled the mtDNA levels in liver compared to the Mpv17 KO, this elevated level still only reached ∼10% of control levels, and no significant improvement in respiratory chain complexes was detected in this tissue. The other devastating aspect of this condition is the short life expectancy, mirrored in the mouse model. With Opa1 overexpression there was a significant change in the median lifespan; however, the maximal lifespan remained almost unchanged from the Mpv17−/− mice. These points do not distract from the evidence that, where there are disorders with clear defects in cristae formation and the cristae junctions, modest overexpression of Opa1 may indeed offer a therapeutic option to alleviate mtDNA depletion or defective mtDNA maintenance. The importance of developing such therapies is self-evident when one considers the ubiquity of mitochondria in all nucleated eukaryotic cells and the important roles they play in a number of different functions, including calcium homeostasis, apoptosis, iron-sulfur cluster formation, and, perhaps most widely recognized, the efficient production of ATP through oxidative phosphorylation (OXPHOS). These reticular organelles are generated and maintained through a coordinated expression of two genomes, with the nuclear DNA contributing approximately 1,200 gene products and the mitochondrial genome encoding 13 open reading frames, all of which are members of the OXPHOS machinery. The latter is positioned within the inner mitochondrial membrane, which is invaginated to form cristae that both increase the surface area of the IMM but, importantly, generates a further discrete intraorganellar subcompartment (see Figure 1). Fission and fusion events determine the reticular or punctate nature of the mitochondrial network. Such rapid adaptation allows the organelle to regulate ATP production and respond to the energetic demands of the cell. These processes of fission and fusion are strictly controlled and involve a number of proteins, including Drp1 (fission), MFN1/2 (fusion), and a further key protein that modulates the cristae architecture, namely Opa1, the potential therapeutic candidate put forward by Luna-Sanchez etal.1Luna-Sanchez M. Benincá C. Cerutti R. Brea-Calvo G. Yeates A. Scorrano L. Zeviani M. Viscomi C. Opa1 Overexpression Protects from Early-Onset Mpv17-/--Related Mouse Kidney Disease.Mol. Ther. 2020; 28 (this issue): 1918-1930Abstract Full Text Full Text PDF Scopus (4) Google Scholar What is becoming increasingly evident is that the balance between fission and fusion coupled with alterations to internal cristae structure serves to regulate and control key aspects of mitochondrial function and that disturbances to this status have implications in development7Wasilewski M. Semenzato M. Rafelski S.M. Robbins J. Bakardjiev A.I. Scorrano L. Optic atrophy 1-dependent mitochondrial remodeling controls steroidogenesis in trophoblasts.Curr. Biol. 2012; 22: 1228-1234Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar and disease (reviewed in Ma et al.8Ma Y. Wang L. Jia R. The role of mitochondrial dynamics in human cancers.Am. J. Cancer Res. 2020; 10: 1278-1293PubMed Google Scholar and Chrzanowska-Lightowlers and Lightowlers9Chrzanowska-Lightowlers Z.M. Lightowlers R.N. How much does a disrupted mitochondrial network influence neuronal dysfunction?.EMBO Mol. Med. 2019; 11: 11Crossref Scopus (1) Google Scholar). For example, reduced levels of Drp1 in skeletal muscle have been seen in a number of muscle wasting conditions,10Favaro G. Romanello V. Varanita T. Andrea Desbats M. Morbidoni V. Tezze C. Albiero M. Canato M. Gherardi G. De Stefani D. et al.DRP1-mediated mitochondrial shape controls calcium homeostasis and muscle mass.Nat. Commun. 2019; 10: 2576Crossref PubMed Scopus (180) Google Scholar and its expression levels and phosphorylation status have been shown to correlate with the severity of progression in various cancers.11Tsuyoshi H. Orisaka M. Fujita Y. Asare-Werehene M. Tsang B.K. Yoshida Y. Prognostic impact of Dynamin related protein 1 (Drp1) in epithelial ovarian cancer.BMC Cancer. 2020; 20: 467Crossref PubMed Scopus (14) Google Scholar Defects in MFN1/2 can cause a form of Charcot Marie tooth disease (OMIM: 609260)12Züchner S. Mersiyanova I.V. Muglia M. Bissar-Tadmouri N. Rochelle J. Dadali E.L. Zappia M. Nelis E. Patitucci A. Senderek J. et al.Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A.Nat. Genet. 2004; 36: 449-451Crossref PubMed Scopus (1238) Google Scholar and Opa1, as its name implies (optic atrophy 1), has also been linked with neurological disorders and disease. It exists in multiple active and inactive forms that need to be correctly balanced to retain a healthy mitochondrial network.13Caglayan S. Hashim A. Cieslar-Pobuda A. Jensen V. Behringer S. Talug B. Chu D.T. Pecquet C. Rogne M. Brech A. et al.Optic Atrophy 1 Controls Human Neuronal Development by Preventing Aberrant Nuclear DNA Methylation.iScience. 2020; 23: 101154Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar These data emphasize that, to ensure correct development and avoid disease, these key proteins that modulate the mitochondrial reticulum need to be maintained at both the appropriate physiological level and modification status. This understanding has led to investigations that have used different in vitro and in vivo approaches to determine whether, by controlling the fission/fusion status of the network, it is possible to correct any imbalances and implement this as a way to suppress dysfunction and prevent disease.14Humphries B.A. Cutter A.C. Buschhaus J.M. Chen Y.C. Qyli T. Palagama D.S.W. Eckley S. Robison T.H. Bevoor A. Chiang B. et al.Enhanced mitochondrial fission suppresses signaling and metastasis in triple-negative breast cancer.Breast Cancer Res. 2020; 22: 60Crossref PubMed Scopus (27) Google Scholar Opa1’s role in mitochondrial architecture made it a prime candidate for such manipulation. Mouse models generated from crosses of Opa1-overexpressing transgenic mice with KO strains displaying dysfunction in either complex I or complex IV of the respiratory chain showed that increased levels of Opa1 could ameliorate the defect in mitochondrial bioenergetics.5Civiletto G. Varanita T. Cerutti R. Gorletta T. Barbaro S. Marchet S. Lamperti C. Viscomi C. Scorrano L. Zeviani M. Opa1 overexpression ameliorates the phenotype of two mitochondrial disease mouse models.Cell Metab. 2015; 21: 845-854Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar When taken all together, these data support the potential of targeting Opa1 expression as a mitochondrial staple gun to restore defective cristae junction structures in disorders where such architectural aberrations affect either mtDNA replication/transcription or the integration of OXPHOS complexes into the inner mitochondrial membranes. The devastating and progressive nature of mitochondrial diseases is a potent reason to consider evaluating all novel therapeutic approaches that could ameliorate these conditions. R.N.L. and Z.M.A.C.-L. were responsible for writing the original draft, review, and editing of this manuscript. The authors declare that there are no conflicts of interest. This work was supported by The Wellcome Trust (203105/Z/16/Z to R.N.L. and Z.M.A.C.-L.) and EU Horizon2020 MCS (ITN REMIX 721757). Opa1 Overexpression Protects from Early-Onset Mpv17−/−-Related Mouse Kidney DiseaseLuna-Sanchez et al.Molecular TherapyJune 11, 2020In BriefNo treatment is currently available to syndromes associated to mitochondrial DNA instability. We investigated the possibility to correct the kidney disease typical of Mpv17−/− mice by transgenically overexpressing Opa1, the master regulator of mitochondrial cristae shape. Mpv17−/−::Opa1 double recombinant mice showed a marked prolongation of the lifespan, with correction of the proteinuria and of the focal segmental glomerulosclerosis, by reducing apoptosis in podocytes. Accordingly, mitochondrial cristae morphology, largely disrupted in Mpv17−/− glomeruli, was improved in double recombinant animals. Full-Text PDF Open Access

In the canonical process of translation, newly completed proteins escape from the ribosome following cleavage of the ester bond that anchors the polypeptide to the P-site tRNA, after which the ribosome can be recycled to initiate a new round of translation. Not all protein synthesis runs to completion as various factors can impede the progression of ribosomes. Rescuing of stalled ribosomes in mammalian mitochondria, however, does not share the same mechanisms that many bacteria use. The classic method for rescuing bacterial ribosomes is trans-translation. The key components of this system are absent from mammalian mitochondria; however, four members of a translation termination factor family are present, with some evidence of homology to members of a bacterial back-up rescue system. To date, there is no definitive demonstration of any other member of this family functioning in mitoribosome rescue. Here, we provide an overview of the processes and key players of canonical translation termination in both bacteria and mammalian mitochondria, followed by a perspective of the bacterial systems used to rescue stalled ribosomes. We highlight any similarities or differences with the mitochondrial translation release factors, and suggest potential roles for these proteins in ribosome rescue in mammalian mitochondria.

Human mitochondria contain their own genome, mtDNA, that is expressed in the mitochondrial matrix. This genome encodes thirteen vital polypeptides that are components of the multi-subunit complexes that couple oxidative phosphorylation (OXPHOS). The inner mitochondrial membrane that houses these complexes comprises the inner boundary membrane that runs parallel to the outer membrane, infoldings that form the cristae membranes, and the cristae junctions that separate the two. It is in these cristae membranes that the OXPHOS complexes have been shown to reside in various species. The majority of the OXPHOS subunits are nuclear-encoded and must therefore be imported from the cytosol through the outer membrane at contact sites with the inner boundary membrane. As the mitochondrially-encoded components are also integral members of these complexes, where does nascent protein synthesis occur? Transcription, mRNA processing, maturation and at least part of the mitoribosome assembly process occur at the nucleoid and the spatially juxtaposed mitochondrial RNA granules, is protein synthesis also performed at the RNA granules close to these entities, or does it occur distal to these sites ? We have adapted a click chemistry based method, coupled with STED nanoscopy to address these questions. We report that in human cells in culture, within the limits of our methodology, the majority of mitochondrial protein synthesis occurs at the cristae membranes and is spatially separated from the sites of RNA processing and maturation.### Competing Interest StatementThe authors have declared no competing interest.

BACKGROUND:Drugs such as taxanes, epothilones, and vinca alkaloids are widely used in the treatment of breast, ovarian, and lung cancers but come with major side effects such as neuropathy and loss of neutrophils and as single agents have a lack of efficacy. M2I-1 (MAD2 inhibitor-1) has been shown to disrupt the CDC20-MAD2 interaction, and consequently, the assembly of the mitotic checkpoint complex (MCC).RESULTS:We report here that M2I-1 can significantly increase the sensitivity of several cancer cell lines to anti-mitotic drugs, with cell death occurring after a prolonged mitotic arrest. In the presence of nocodazole or taxol combined with M2I-1 cell death is triggered by the premature degradation of Cyclin B1, the perturbation of the microtubule network, and an increase in the level of the pro-apoptotic protein MCL-1s combined with a marginal increase in the level of NOXA. The elevated level of MCL-1s and the marginally increased NOXA antagonized the increased level of MCL-1, a pro-survival protein of the Bcl-2 family.CONCLUSION:Our results provide some important molecular mechanisms for understanding the relationship between the mitotic checkpoint and programmed cell death and demonstrate that M2I-1 exhibits antitumor activity in the presence of current anti-mitotic drugs such as taxol and nocodazole and has the potential to be developed as an anticancer agent.

Mitochondria are ubiquitous organelles present in the cytoplasm of all nucleated eukaryotic cells. These organelles are described as arising from a common ancestor but a comparison of numerous aspects of mitochondria between different organisms provides remarkable examples of divergent evolution. In humans, these organelles are of dual genetic origin, comprising ∼1500 nuclear-encoded proteins and thirteen that are encoded by the mitochondrial genome. Of the various functions that these organelles perform, it is only oxidative phosphorylation, which provides ATP as a source of chemical energy, that is dependent on synthesis of these thirteen mitochondrially encoded proteins. A prerequisite for this process of translation are the mitoribosomes. The recent revolution in cryo-electron microscopy has generated high-resolution mitoribosome structures and has undoubtedly revealed some of the most distinctive molecular aspects of the mitoribosomes from different organisms. However, we still lack a complete understanding of the mechanistic aspects of this process and many of the factors involved in post-transcriptional gene expression in mitochondria. This review reflects on the current knowledge and illustrates some of the striking differences that have been identified between mitochondria from a range of organisms.