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Mitochondria rely on coordinated expression of their own mitochondrial DNA (mtDNA) with that of the nuclear genome for their biogenesis. The bacterial ancestry of mitochondria has given rise to unique and idiosyncratic features of the mtDNA and its expression machinery that can be specific to different organisms. In animals, the mitochondrial protein synthesis machinery has acquired many new components and mechanisms over evolution. These include several new ribosomal proteins, new stop codons and ways to recognise them, and new mechanisms to deliver nascent proteins into the mitochondrial inner membrane. Here we describe the mitochondrial protein synthesis machinery in mammals and its unique mechanisms of action elucidated to date and highlight the technologies poised to reveal the next generation of discoveries in mitochondrial translation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development., (© The Author(s) 2024. Published by Oxford University Press.)

The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.

Changes in the rate and fidelity of mitochondrial protein synthesis impact the metabolic and physiological roles of mitochondria. Here we explored how environmental stress in the form of a high-fat diet modulates mitochondrial translation and affects lifespan in mutant mice with error-prone (Mrps12 ep / ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12 ep ) or hyper-accurate (Mrps12 ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging./ ha ) mitochondrial ribosomes. Intriguingly, although both mutations are metabolically beneficial in reducing body weight, decreasing circulating insulin and increasing glucose tolerance during a high-fat diet, they manifest divergent (either deleterious or beneficial) outcomes in a tissue-specific manner. In two distinct organs that are commonly affected by the metabolic disease, the heart and the liver, Mrps12 ep / ep mice were protected against heart defects but sensitive towards lipid accumulation in the liver, activating genes involved in steroid and amino acid metabolism. In contrast, enhanced translational accuracy in Mrps12 ha / ha mice protected the liver from a high-fat diet through activation of liver proliferation programs, but enhanced the development of severe hypertrophic cardiomyopathy and led to reduced lifespan. These findings reflect the complex transcriptional and cell signalling responses that differ between post-mitotic (heart) and highly proliferative (liver) tissues. We show trade-offs between the rate and fidelity of mitochondrial protein synthesis dictate tissue-specific outcomes due to commonly encountered stressful environmental conditions or aging., (© 2021 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Transcriptomic technologies have revolutionized the study of gene expression and RNA biology. Different RNA sequencing methods enable the analyses of diverse species of transcripts, including their abundance, processing, stability, and other specific features. Mitochondrial transcriptomics has benefited from these technologies that have revealed the surprising complexity of its RNAs. Here we describe a method based upon cyclization of mitochondrial RNAs and next generation sequencing to analyze the steady-state levels and sizes of mitochondrial RNAs, their degradation products, as well as their processing intermediates by capturing both 5' and 3' ends of transcripts.

Translation fidelity is crucial for prokaryotes and eukaryotic nuclear-encoded proteins; however, little is known about the role of mistranslation in mitochondria and its potential effects on metabolism. We generated yeast and mouse models with error-prone and hyper-accurate mitochondrial translation, and found that translation rate is more important than translational accuracy for cell function in mammals. Specifically, we found that mitochondrial mistranslation causes reduced overall mitochondrial translation and respiratory complex assembly rates. In mammals, this effect is compensated for by increased mitochondrial protein stability and upregulation of the citric acid cycle. Moreover, this induced mitochondrial stress signaling, which enables the recovery of mitochondrial translation via mitochondrial biogenesis, telomerase expression, and cell proliferation, and thereby normalizes metabolism. Conversely, we show that increased fidelity of mitochondrial translation reduces the rate of protein synthesis without eliciting a mitochondrial stress response. Consequently, the rate of translation cannot be recovered and this leads to dilated cardiomyopathy in mice. In summary, our findings reveal mammalian-specific signaling pathways that respond to changes in the fidelity of mitochondrial protein synthesis and affect metabolism., (© 2019 The Authors.)

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length and are assumed to constitute the final stage of RNA turnover. Here we demonstrate that REXO2 is a specialized dinucleotide-degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. A heart- and skeletal-muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro. Our data demonstrate that increased levels of dinucleotides can be used to initiate transcription, leading to an increase in transcription levels from both mitochondrial promoters and other, nonspecific sequence elements in mitochondrial DNA. Efficient RNA turnover by REXO2 is thus required to maintain promoter specificity and proper regulation of transcription in mammalian mitochondria., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondrial transfer plays a crucial role in the regulation of tissue homeostasis and resistance to cancer chemotherapy. Osteocytes have interconnecting dendritic networks and are a model to investigate its mechanism. We have demonstrated, in primary murine osteocytes with photoactivatable mitochondria (PhAM) floxed and in MLO-Y4 cells, mitochondrial transfer in the dendritic networks visualized by high-resolution confocal imaging. Normal osteocytes transferred mitochondria to adjacent metabolically stressed osteocytes and restored their metabolic function. The coordinated movement and transfer of mitochondria within the dendritic network rely on contact between the endoplasmic reticulum (ER) and mitochondria. Mitofusin 2 (Mfn2), a GTPase that tethers ER to mitochondria, predominantly mediates the transfer. A decline in Mfn2 expression with age occurs concomitantly with both impaired mitochondrial distribution and transfer in the osteocyte dendritic network. These data show a previously unknown function of ER-mitochondrial contact in mediating mitochondrial transfer and provide a mechanism to explain the homeostasis of osteocytes., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm -knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria., (© 2019 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Mitochondrial biogenesis is intimately dependent on the coordinated expression of the nuclear and mitochondrial genomes that is necessary for the assembly and function of the respiratory complexes to produce most of the energy required by cells. Although highly compacted in animals, the mitochondrial genome and its expression are essential for survival, development, and optimal energy production. The machinery that regulates gene expression within mitochondria is localised within the same compartment and, like in their ancestors, the bacteria, this machinery does not use membrane-based compartmentalisation to order the gene expression pathway. Therefore, the lifecycle of mitochondrial RNAs from transcription through processing, maturation, translation to turnover is mediated by a gamut of RNA-binding proteins (RBPs), all contained within the mitochondrial matrix milieu. Recent discoveries indicate that multiple processes regulating RNA metabolism occur at once but since mitochondria have a new complement of RBPs, many evolved de novo from nuclear genes, we are left wondering how co-ordinated are these processes? Here, we review recently identified examples of the co-ordinated and stochastic processes that govern the mitochondrial transcriptome. These new discoveries reveal the complexity of mitochondrial gene expression and the need for its in-depth exploration to understand how these organelles can respond to the energy demands of the cell., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation 1,2 . The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals 3,4 . Furthermore, only one type of methionyl transfer RNA (tRNA Met ) is used for both initiation and elongation 4,5 , necessitating that the initiation factor specifically recognizes the formylated version of tRNA Met (fMet-tRNA Met ). Lastly, most mitochondrial mRNAs do not possess 5' leader sequences to promote mRNA binding to the ribosome 2 . There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNA Met and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane.

The expression of the compact mammalian mitochondrial genome requires transcription, RNA processing, translation and RNA decay, much like the more complex chromosomal systems, and here we use it as a model system to understand the fundamental aspects of gene expression. Here we combine RNase footprinting with PAR-CLIP at unprecedented depth to reveal the importance of RNA-protein interactions in dictating RNA folding within the mitochondrial transcriptome. We show that LRPPRC, in complex with its protein partner SLIRP, binds throughout the mitochondrial transcriptome, with a preference for mRNAs, and its loss affects the entire secondary structure and stability of the transcriptome. We demonstrate that the LRPPRC-SLIRP complex is a global RNA chaperone that stabilizes RNA structures to expose the required sites for translation, stabilization, and polyadenylation. Our findings reveal a general mechanism where extensive RNA-protein interactions ensure that RNA is accessible for its biological functions.

Dysfunction of the oxidative phosphorylation (OXPHOS) system is a major cause of human disease and the cellular consequences are highly complex. Here, we present comparative analyses of mitochondrial proteomes, cellular transcriptomes and targeted metabolomics of five knockout mouse strains deficient in essential factors required for mitochondrial DNA gene expression, leading to OXPHOS dysfunction. Moreover, we describe sequential protein changes during post-natal development and progressive OXPHOS dysfunction in time course analyses in control mice and a middle lifespan knockout, respectively. Very unexpectedly, we identify a new response pathway to OXPHOS dysfunction in which the intra-mitochondrial synthesis of coenzyme Q (ubiquinone, Q) and Q levels are profoundly decreased, pointing towards novel possibilities for therapy. Our extensive omics analyses provide a high-quality resource of altered gene expression patterns under severe OXPHOS deficiency comparing several mouse models, that will deepen our understanding, open avenues for research and provide an important reference for diagnosis and treatment.

The central role of the mitochondrion for cellular and organismal metabolism is well known, yet its functional role in evolution has rarely been featured in leading international conferences. Moreover, the contribution of mitochondrial genetics to complex disease phenotypes is particularly important, and although major advances have been made in the field of genomics, mitochondrial genomic data have in many cases been overlooked. Accumulating data and new knowledge support a major contribution of this maternally inherited genome, and its interactions with the nucleus, to both major evolutionary processes and diverse disease phenotypes. These advances encouraged us to assemble the first Mitochondrial Genomics and Evolution (MGE) meeting-an SMBE satellite and Israeli Science foundation international conference (Israel, September 2017). Here, we report the content and outcome of the MGE meeting (https://www.mge2017.com/; last accessed November 5, 2017)., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The recognition and translation of mammalian mitochondrial mRNAs are poorly understood. To gain further insights into these processes in vivo, we characterized mice with a missense mutation that causes loss of the translational activator of cytochrome oxidase subunit I (TACO1). We report that TACO1 is not required for embryonic survival, although the mutant mice have substantially reduced COXI protein, causing an isolated complex IV deficiency. We show that TACO1 specifically binds the mt-Co1 mRNA and is required for translation of COXI through its association with the mitochondrial ribosome. We determined the atomic structure of TACO1, revealing three domains in the shape of a hook with a tunnel between domains 1 and 3. Mutations in the positively charged domain 1 reduce RNA binding by TACO1. The Taco1 mutant mice develop a late-onset visual impairment, motor dysfunction and cardiac hypertrophy and thus provide a useful model for future treatment trials for mitochondrial disease.

Leigh syndrome (LS) is a progressive mitochondrial neurodegenerative disorder, whose symptoms most commonly include psychomotor delay with regression, lactic acidosis and a failure to thrive. Here we describe three siblings with LS, but with additional manifestations including hypertrophic cardiomyopathy, hepatosplenomegaly, cholestatic hepatitis, and seizures. All three affected siblings were found to be homoplasmic for an m. 5559A>G mutation in the T stem of the mitochondrial DNA-encoded MT-TW by next generation sequencing. The m.5559A>G mutation causes a reduction in the steady state levels of tRNA(Trp) and this decrease likely affects the stability of other mitochondrial RNAs in the patient fibroblasts. We observe accumulation of an unprocessed transcript containing tRNA(Trp), decreased de novo protein synthesis and consequently lowered steady state levels of mitochondrial DNA-encoded proteins that compromise mitochondrial respiration. Our results show that the m.5559A>G mutation at homoplasmic levels causes LS in association with severe multi-organ disease (LS-plus) as a consequence of dysfunctional mitochondrial RNA metabolism., (Copyright © 2015 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.

Estrogens, in particular 17β-estradiol, are well-known regulators of essential cellular functions; however, discrepancies remain over the mechanisms by which they act on mitochondria. Here we propose a novel mechanism for the direct regulation of mitochondrial gene expression by estrogen under metabolic stress. We show that in serum-depleted medium, estrogen stimulates a rapid relocation of estrogen receptor-α to mitochondria, in which it elicits a cellular response, resulting in an increase in mitochondrial RNA abundance. Mitochondrial RNA levels are regulated through the association of estrogen receptor-α with 17β-hydroxysteroid dehydrogenase 10, a multifunctional protein involved in steroid metabolism that is also a core subunit of the mitochondrial ribonuclease P complex responsible for the cleavage of mitochondrial polycistronic transcripts. Processing of mitochondrial transcripts affects mitochondrial gene expression by controlling the levels of mature RNAs available for translation. This work provides the first mechanism linking RNA processing and estrogen activation in mitochondrial gene expression and underscores the coordinated response between the nucleus and mitochondria in response to stress.

Mitochondria are responsible for generating most of the energy required by the cell. The oxidative phosphorylation (OXPHOS) system that produces the energy is composed of nuclear and mitochondrial encoded polypeptides. The 13 polypeptides encoded by the mitochondrial genome are synthesized by mitochondrial ribosomes (mitoribosomes). The evolutionary divergence of mitoribosomes has seen a reduction in their rRNA content and an increase in ribosomal proteins compared to their bacterial and cytoplasmic counterparts. Recent advances in cryo-electron microscopy (cryo-EM) mapping have revealed not all of these proteins simply replace the roles of the rRNA and that many have new roles. The mitoribosome has unique features that include a gatelike structure at the mRNA entrance that may facilitate recruitment of leaderless mitochondrial mRNAs and also a polypeptide exit tunnel that has an unusual nascent-polypeptide exit mechanism. Defects in the mitochondrial translation machinery are a common contributor to multi-system disorders known as mitochondrial diseases for which currently there are no cures or effective treatments., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Messenger RNAs encoded by mitochondrial genomes are translated on mitochondrial ribosomes that have unique structure and protein composition compared to prokaryotic and cytoplasmic ribosomes. Mitochondrial ribosomes are a patchwork of core proteins that share homology with prokaryotic ribosomal proteins and new, supernumerary proteins that can be unique to different organisms. In mammals, there are specific supernumerary ribosomal proteins that are not present in other eukaryotes., Scope of Review: Here we discuss the roles of supernumerary proteins in the regulation of mitochondrial gene expression and compare them among different eukaryotic systems. Furthermore, we consider if differences in the structure and organization of mitochondrial genomes may have contributed to the acquisition of mitochondrial ribosomal proteins with new functions., Major Conclusions: The distinct and diverse compositions of mitochondrial ribosomes illustrate the high evolutionary divergence found between mitochondrial genetic systems., General Significance: Elucidating the role of the organism-specific supernumerary proteins may provide a window into the regulation of mitochondrial gene expression through evolution in response to distinct evolutionary paths taken by mitochondria in different organisms. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research., (© 2013.)

Genetic drift and mutational pressure have shaped the evolution of mitochondrial and chloroplast genomes, giving rise to mechanisms that regulate their gene expression, which often differ from those in their prokaryotic ancestors. Advances in next generation sequencing technologies have enabled highly detailed characterization of organelle transcriptomes and the discovery of new transcripts and mechanisms for controlling gene expression. Here we discuss the common features of organelle transcriptomes that stem from their prokaryotic origin and some of the new innovations that are unique to organelles of multicellular organisms., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Pentatricopeptide repeat (PPR) domain proteins are a large family of RNA-binding proteins that are involved in the maturation and translation of organelle transcripts in eukaryotes. They were first identified in plant organelles and their important role in mammalian mitochondrial gene regulation is now emerging. Mammalian PPR proteins, like their plant counterparts, have diverse roles in mitochondrial transcription, RNA metabolism and translation and consequently are important for mitochondrial function and cell health. Here we discuss the current knowledge about the seven mammalian PPR proteins identified to date and their roles in the regulation of mitochondrial gene expression. Furthermore we discuss the mitochondrial RNA targets of the mammalian PPR proteins and methods to investigate the RNA targets of these mitochondrial RNA-binding proteins. This article is part of a Special Issue entitled: Mitochondrial Gene Expression., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The human mitochondrial transcriptome, although produced from a small and compact genome, has revealed surprising complexity in its composition and regulation. Wide variation between individual tRNAs, mRNAs, and rRNAs indicate the importance of post-transcriptional processing, maturation, and degradation mechanisms in the regulation of mitochondrial gene expression. RNA-binding proteins play essential roles in controlling the mitochondrial transcriptome from its synthesis to its destruction and have evolved unique features to complement the unusual features of mitochondrial RNAs. Recent studies have shown how changes in mitochondrial RNAs and their binding proteins can have significant effects on human health. This opens new avenues for investigation of mitochondrial RNA-binding proteins and the mechanisms by which they regulate mitochondrial gene expression., (Copyright © 2012 John Wiley & Sons, Ltd.)

Mammalian mitochondrial DNA is transcribed as precursor polycistronic transcripts containing 13 mRNAs, 2 rRNAs, punctuated by 22 tRNAs. The mechanisms involved in the excision of mitochondrial tRNAs from these polycistronic transcripts have remained largely unknown. We have investigated the roles of ELAC2, mitochondrial RNase P proteins 1 and 3, and pentatricopeptide repeat domain protein 1 in the processing of mitochondrial polycistronic transcripts. We used a deep sequencing approach to characterize the 5' and 3' ends of processed mitochondrial transcripts and provide a detailed map of mitochondrial tRNA processing sites affected by these proteins. We show that MRPP1 and MRPP3 process the 5' ends of tRNAs and the 5' unconventional, non tRNA containing site of the CO1 transcript. By contrast, we find that ELAC2 and PTCD1 affect the 3' end processing of tRNAs. Finally, we found that MRPP1 is essential for transcript processing, RNA modification, translation and mitochondrial respiration., (© 2011 Landes Bioscience)

The human mitochondrial genome comprises a distinct genetic system transcribed as precursor polycistronic transcripts that are subsequently cleaved to generate individual mRNAs, tRNAs, and rRNAs. Here, we provide a comprehensive analysis of the human mitochondrial transcriptome across multiple cell lines and tissues. Using directional deep sequencing and parallel analysis of RNA ends, we demonstrate wide variation in mitochondrial transcript abundance and precisely resolve transcript processing and maturation events. We identify previously undescribed transcripts, including small RNAs, and observe the enrichment of several nuclear RNAs in mitochondria. Using high-throughput in vivo DNaseI footprinting, we establish the global profile of DNA-binding protein occupancy across the mitochondrial genome at single-nucleotide resolution, revealing regulatory features at mitochondrial transcription initiation sites and functional insights into disease-associated variants. This integrated analysis of the mitochondrial transcriptome reveals unexpected complexity in the regulation, expression, and processing of mitochondrial RNA and provides a resource for future studies of mitochondrial function (accessed at http://mitochondria.matticklab.com)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The disulfide relay system of the mitochondrial intermembrane space has been extensively characterized in Saccharomyces cerevisiae. It contains two essential components, Mia40 and Erv1. The genome of Arabidopsis thaliana contains a single gene for each of these components. Although insertional inactivation of Erv1 leads to a lethal phenotype, inactivation of Mia40 results in no detectable deleterious phenotype. A. thaliana Mia40 is targeted to and accumulates in mitochondria and peroxisomes. Inactivation of Mia40 results in an alteration of several proteins in mitochondria, an absence of copper/zinc superoxide dismutase (CSD1), the chaperone for superoxide dismutase (Ccs1) that inserts copper into CSD1, and a decrease in capacity and amount of complex I. In peroxisomes the absence of Mia40 leads to an absence of CSD3 and a decrease in abnormal inflorescence meristem 1 (Aim1), a β-oxidation pathway enzyme. Inactivation of Mia40 leads to an alteration of the transcriptome of A. thaliana, with genes encoding peroxisomal proteins, redox functions, and biotic stress significantly changing in abundance. Thus, the mechanistic operation of the mitochondrial disulfide relay system is different in A. thaliana compared with other systems, and Mia40 has taken on new roles in peroxisomes and mitochondria.

The basic components and mechanisms of mitochondrial transcription in mammals have been described, however, the components involved in mRNA processing, translation and stability remain largely unknown. In plants, pentatricopeptide domain RNA-binding proteins regulate the stability, expression and translation of mitochondrial transcripts. Here, we investigated the role of an uncharacterized mammalian pentatricopeptide domain protein, pentatricopeptide repeat domain protein 3 (PTCD3), and showed that it is a mitochondrial protein that associates with the small subunit of mitochondrial ribosomes. PTCD3 knockdown and over expression did not affect mitochondrial mRNA levels, suggesting that PTCD3 is not involved in RNA processing and stability. However, lowering PTCD3 in 143B osteosarcoma cells decreased mitochondrial protein synthesis, mitochondrial respiration and the activity of Complexes III and IV, suggesting that PTCD3 has a role in mitochondrial translation.

Mitochondrial oxidative damage is thought to contribute to a wide range of human diseases; therefore, the development of approaches to decrease this damage may have therapeutic potential. Mitochondria-targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death have been developed. These compounds contain antioxidant moieties, such as ubiquinone, tocopherol, or nitroxide, that are targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondrial membrane potential, the cations are accumulated within the mitochondria inside cells. There, the conjugated antioxidant moiety protects mitochondria from oxidative damage. Here, we outline some of the work done to date on these compounds and how they may be developed as therapies.

A family of lipophilic, cationic Au(I) complexes of N-heterocyclic carbenes (NHCs) have been designed as new mitochondria-targeted antitumor agents that combine both selective mitochondrial accumulation and selective thioredoxin reductase inhibition properties within a single molecule. Two-step ligand exchange reactions with cysteine (Cys) and selenocysteine (Sec) occur with release of the NHC ligands. At physiological pH the rate constants for the reactions with Sec are 20- to 80-fold higher than those with Cys. The complexes are selectively toxic to two highly tumorigenic breast cancer cell lines and not to normal breast cells, and the degree of selectivity and potency are optimized by modification of the substituent on the simple imidazolium salt precursor. The lead compound is shown to accumulate in mitochondria of cancer cells, to cause cell death through a mitochondrial apoptotic pathway and to inhibit the activity of thioredoxin reductase (TrxR) but not the closely related and Se-free enzyme glutathione reductase.

A hallmark of cancer cells is their ability to evade apoptosis and mitochondria play a critical role in this process. Delineating mitochondrial differences between normal and cancer cells has proven challenging due to the lack of matched cell lines. Here, we compare two matched liver progenitor cell (LPC) lines, one non-tumorigenic [p53-immortalized liver (PIL) 4] and the other tumorigenic (PIL2). Analysis of these cell lines and a p53 wild-type non-tumorigenic cell line [bipotential murine oval liver (BMOL)] revealed an increase in expression of genes encoding the antiapoptotic proteins cellular inhibitor of apoptosis protein (cIAP) 1 and yes associate protein in the PIL2 cells, which resulted in an increase in the protein encoded by these genes. PIL2 cells have higher mitochondrial membrane potential (Deltapsi(m)) compared with PIL4 and BMOL and had greater levels of reactive oxygen species, despite the fact that the mitochondrial antioxidant enzyme, manganese superoxide disumutase, was elevated at transcript and protein levels. Taken together, these results may account for the observed resistance of PIL2 cells to apoptotic stimuli compared with PIL4. We tested a new gold compound to show that hyperpolarized Deltapsi(m) led to its increased accumulation in mitochondria of PIL2 cells. This compound selectively induces apoptosis in PIL2 cells but not in PIL4 or BMOL. The gold compound depolarized the Deltapsi(m), depleted the adenosine triphosphate pool and activated caspase-3 and caspase-9, suggesting that apoptosis was mediated via mitochondria. This investigation shows that the non-tumorigenic and tumorigenic LPCs are useful models to delineate the role of mitochondrial dysfunction in tumorigenesis and for the future development of mitochondria-targeted chemotherapeutics that selectively target tumor cells.

A large number of proteins contain free thiols that can be modified by the formation of internal disulphides or by mixed disulphides with low-molecular-mass thiols. The majority of these latter modifications result from the interaction of protein thiols with the endogenous glutathione pool. Protein glutathionylation and disulphide formation are of significance both for defence against oxidative damage and in redox signalling. As mitochondria are central to both oxidative damage and redox signalling within the cell, these modifications of mitochondrial proteins are of particular importance. In the present study, we review the mechanisms and physiological significance of these processes.

Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.

CPPs (cell-penetrating peptides) facilitate the cellular uptake of covalently attached oligonucleotides, proteins and other macromolecules, but the mechanism of their uptake is disputed. Two models are proposed: direct movement through the phospholipid bilayer and endocytic uptake. Mitochondria are a good model system to distinguish between these possibilities, since they have no vesicular transport systems. Furthermore, CPP-mediated delivery of macromolecules to the mitochondrial matrix would be a significant breakthrough in the study of mitochondrial function and dysfunction, and could also lead to new therapies for diseases caused by mitochondrial damage. Therefore we investigated whether two CPPs, penetratin and Tat, could act as mitochondrial delivery vectors. We also determined whether conjugation of the lipophilic cation TPP (triphenylphosphonium) to penetratin or Tat facilitated their uptake into mitochondria, since TPP leads to uptake of attached molecules into mitochondria driven by the membrane potential. Neither penetratin nor Tat, nor their TPP conjugates, are internalized by isolated mitochondria, indicating that these CPPs cannot cross mitochondrial phospholipid bilayers. Tat and TPP-Tat are taken up by cells, but they accumulate in endosomes and do not reach mitochondria. We conclude that CPPs cannot cross mitochondrial phospholipid bilayers, and therefore cannot deliver macromolecules directly to mitochondria. Our findings shed light on the mechanism of uptake of CPPs by cells. The lack of direct movement of CPPs through mitochondrial phospholipid bilayers, along with the observed endosomal accumulation of Tat and TPP-Tat in cells, makes it unlikely that CPPs enter cells by direct membrane passage, and instead favours cellular uptake via an endocytic pathway.

The production of ROS (reactive oxygen species) by the mitochondrial respiratory chain contributes to a range of pathologies, including neurodegenerative diseases, ischaemia/reperfusion injury and aging. There are also indications that mitochondrial ROS production plays a role in damage response and signal transduction pathways. To unravel the role of mitochondrial ROS production in these processes, we have developed a range of mitochondria-targeted probe molecules. Covalent attachment of a lipophilic cation leads to their accumulation into mitochondria, driven by the membrane potential. Molecules developed so far include antioxidants designed to intercept mitochondrial ROS and reagents that specifically label mitochondrial thiol proteins. Here we outline how mitochondrial ROS formation and its consequences can be investigated using these probes.

Abstract Prostate cancer is the most commonly diagnosed malignancy and the third leading cause of cancer deaths. GWAS have identified variants associated with prostate cancer susceptibility; however, mechanistic and functional validation of these mutations is lacking. We used CRISPR‐Cas9 genome editing to introduce a missense variant identified in the ELAC2 gene, which encodes a dually localised nuclear and mitochondrial RNA processing enzyme, into the mouse Elac2 gene as well as to generate a prostate‐specific knockout of Elac2. These mutations caused enlargement and inflammation of the prostate and nodule formation. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer. Multiomic profiling revealed defects in energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired noncoding RNA processing and reduced protein synthesis. Our physiologically relevant models show that the ELAC2 variant is a predisposing factor for prostate cancer and identify changes that underlie the pathogenesis of this cancer.

The regulation of mitochondrial RNA processing and its importance for ribosome biogenesis and energy metabolism are not clear. We generated conditional knockout mice of the endoribonuclease component of the RNase P complex, MRPP3, and report that it is essential for life and that heart and skeletal-muscle-specific knockout leads to severe cardiomyopathy, indicating that its activity is non-redundant. Transcriptome-wide parallel analyses of RNA ends (PARE) and RNA-seq enabled us to identify that in vivo 5′ tRNA cleavage precedes 3′ tRNA processing, and this is required for the correct biogenesis of the mitochondrial ribosomal subunits. We identify that mitoribosomal biogenesis proceeds co-transcriptionally because large mitoribosomal proteins can form a subcomplex on an unprocessed RNA containing the 16S rRNA. Taken together, our data show that RNA processing links transcription to translation via assembly of the mitoribosome.

Heterozygous mice (αMHC403/+) expressing the human disease-causing mutation Arg403Gln exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+. These increases were attenuated with ICa-L antagonists and following F-actin or β-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the Arg403Gln mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.

Prostate cancer is the most commonly diagnosed malignancy and the third leading cause of cancer deaths. GWAS have identified variants associated with prostate cancer susceptibility; however, mechanistic and functional validation of these mutations is lacking. We used CRISPR‐Cas9 genome editing to introduce a missense variant identified in the ELAC2 gene, which encodes a dually localised nuclear and mitochondrial RNA processing enzyme, into the mouse Elac2 gene as well as to generate a prostate‐specific knockout of Elac2. These mutations caused enlargement and inflammation of the prostate and nodule formation. The Elac2 variant or knockout mice on the background of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model show that Elac2 mutation with a secondary genetic insult exacerbated the onset and progression of prostate cancer. Multiomic profiling revealed defects in energy metabolism that activated proinflammatory and tumorigenic pathways as a consequence of impaired noncoding RNA processing and reduced protein synthesis. Our physiologically relevant models show that the ELAC2 variant is a predisposing factor for prostate cancer and identify changes that underlie the pathogenesis of this cancer. Synopsis: Polymorphisms in ELAC2 have been correlated with prostate cancer, but a definitive link has been lacking. Introducing a common prostate‐susceptibility variant into mice increases inflammation, perturbs the production of non‐coding RNAs, and rewires cellular signaling to exacerbate tumorigenesis. Mice carrying a point mutation equivalent to the human Ala541Thr variant in ELAC2 have inflammation of the prostate and nodule formation.In the presence of a secondary oncogenic stimulus, the ELAC2 variant exacerbates prostate cancer onset and progression.The ELAC2 polymorphism increases cell proliferation, migration, immunometabolic and inflammatory pathways, such as MAPK activation.Production of noncoding RNAs, including tRNAs and miRNAs, is perturbed, leading to defects in protein homeostasis and OXPHOS.ELAC2 interacts with NONO/SFPQ, and the ELAC2 polymorphism reduces this interaction, providing an additional link between altered gene expression in the nucleus and mitochondria. [ABSTRACT FROM AUTHOR]

Canonical RNA processing in mammalian mitochondria is defined by tRNAs acting as recognition sites for nucleases to release flanking transcripts. The relevant factors, their structures, and mechanism are well described, but not all mitochondrial transcripts are punctuated by tRNAs, and their mode of processing has remained unsolved. Using Drosophila and mouse models, we demonstrate that non-canonical processing results in the formation of 3′ phosphates, and that phosphatase activity by the carbon catabolite repressor 4 domain-containing family member ANGEL2 is required for their hydrolysis. Furthermore, our data suggest that members of the FAST kinase domain-containing protein family are responsible for these 3′ phosphates. Our results therefore propose a mechanism for non-canonical RNA processing in metazoan mitochondria, by identifying the role of ANGEL2. A subset of mitochondrial transcripts is not flanked by tRNAs and thus does not conform to the canonical mode of processing. Here, Clemente et al. demonstrate that phosphatase activity of ANGEL2 is required for correct processing of these transcripts. [ABSTRACT FROM AUTHOR]