Mitochondrial Transcription and Translation: Overview

Mitochondria are the major source of ATP in the cell. Five multi-subunit complexes in the inner membrane of the organelle are involved in the oxidative phosphorylation required for ATP production. Thirteen subunits of these complexes are encoded by the mitochondrial genome often referred to as mtDNA. For this reason, the expression of mtDNA is vital for the assembly and functioning of the oxidative phosphorylation complexes. Defects of the mechanisms regulating mtDNA gene expression have been associated with deficiencies in assembly of these complexes, resulting in mitochondrial diseases. Recently, numerous factors involved in these processes have been identified and characterised leading to a deeper understanding of the mechanisms that underlie mitochondrial diseases

Human mitochondrial ribosomes can switch structural tRNAs - but when and why?

High resolution cryoEM of mammalian mitoribosomes revealed the unexpected presence of mitochondrially encoded tRNA as a structural component of mitochondrial large ribosomal subunit (mt-LSU). Our previously published data identified that only mitochondrial (mt-) tRNA(Phe) and mt-tRNA(Val) can be incorporated into mammalian mt-LSU and within an organism there is no evidence of tissue specific variation. When mt-tRNA(Val) is limiting, human mitoribosomes can integrate mt-tRNA(Phe) instead to generate a translationally competent monosome. Here we discuss the possible reasons for and consequences of the observed plasticity of the structural mt-tRNA integration. We also indicate potential direction for further research that could help our understanding of the mechanistic and evolutionary aspects of this unprecedented system.This work was supported by the Wellcome Trust under Grant 096919/Z/11/Z and Medical Research Council UK under Grant MC_U105697135

Gene therapy for mitochondrial disorders

In this review, we detail the current state of application of gene therapy to primary mitochondrial disorders (PMDs). Recombinant adeno-associated virus-based (rAAV) gene replacement approaches for nuclear gene disorders have been undertaken successfully in more than ten preclinical mouse models of PMDs which has been made possible by the development of novel rAAV technologies that achieve more efficient organ targeting. So far, however, the greatest progress has been made for Leber Hereditary Optic Neuropathy, for which phase 3 clinical trials of lenadogene nolparvovec demonstrated efficacy and good tolerability. Other methods of treating mitochondrial DNA (mtDNA) disorders have also had traction, including refinements to nucleases that degrade mtDNA molecules with pathogenic variants, including transcription activator-like effector nucleases, zinc-finger nucleases, and meganucleases (mitoARCUS). rAAV-based approaches have been used successfully to deliver these nucleases in vivo in mice. Exciting developments in CRISPR-Cas9 gene editing technology have achieved in vivo gene editing in mouse models of PMDs due to nuclear gene defects and new CRISPR-free gene editing approaches have shown great potential for therapeutic application in mtDNA disorders. We conclude the review by discussing the challenges of translating gene therapy in patients both from the point of view of achieving adequate organ transduction as well as clinical trial design

Macropinocytotic entry of isolated mitochondria in epidermal growth factor-activated human osteosarcoma cells

Mammalian mitochondria can be transferred between cells both in culture and in vivo. There is evidence that isolated mitochondria enter cells by endocytosis, but the mechanism has not been fully characterised. We investigated the entry mechanism of isolated mitochondria into human osteosarcoma (HOS) cells. Initially we confirmed that respiratory-competent cells can be produced following incubation of HOS cells lacking mitochondrial DNA (mtDNA) with functional exogenous mitochondria and selection in a restrictive medium. Treatment of HOS cells with inhibitors of different endocytic pathways suggest that uptake of EGFP-labelled mitochondria occurs via an actin-dependent endocytic pathway which is consistent with macropinocytosis. We later utilised time-lapse microscopy to show that internalised mitochondria were found in large, motile cellular vesicles. Finally, we used confocal imaging to show that EGFP-labelled mitochondria colocalise with a macropinocytic cargo molecule during internalisation, HOS cells produce membrane ruffles interacting with external mitochondria during uptake and EGFP-labelled mitochondria are found within early macropinosomes inside cells. In conclusion our results are consistent with isolated mitochondria being internalised by macropinocytosis in HOS cells.This work was supported by a grant from the Medical Research Council UK (MC_U105697135). We are grateful to Folma Buss for very helpful discussions during the course of this wo

Localisation of the human hSuv3p helicase in the mitochondrial matrix and its preferential unwinding of dsDNA

We characterised the human hSuv3p protein belonging to the family of NTPases/helicases. In yeast mitochondria the hSUV3 orthologue is a component of the degradosome complex and participates in mtRNA turnover and processing, while in Caenorhabditis elegans the hSUV3 orthologue is necessary for viability of early embryos. Using immunofluorescence analysis, an in vitro mitochondrial uptake assay and sub‐fractionation of human mitochondria we show hSuv3p to be a soluble protein localised in the mitochondrial matrix. We expressed and purified recombinant hSuv3p protein from a bacterial expression system. The purified enzyme was capable of hydrolysing ATP with a Km of 41.9 µM and the activity was only modestly stimulated by polynucleotides. hSuv3p unwound partly hybridised dsRNA and dsDNA structures with a very strong preference for the latter. The presented analysis of the hSuv3p NTPase/helicase suggests that new functions of the protein have been acquired in the course of evolution

Therapeutic Manipulation of mtDNA Heteroplasmy : A Shifting Perspective

Mutations of mitochondrial DNA (mtDNA) often underlie mitochondrial disease, one of the most common inherited metabolic disorders. Since the sequencing of the human mitochondrial genome and the discovery of pathogenic mutations in mtDNA more than 30 years ago, a movement towards generating methods for robust manipulation of mtDNA has ensued, although with relatively few advances and some controversy. While developments in the transformation of mammalian mtDNA have stood still for some time, recent demonstrations of programmable nuclease-based technology suggest that clinical manipulation of mtDNA heteroplasmy may be on the horizon for these largely untreatable disorders. Here we review historical and recent developments in mitochondrially targeted nuclease technology and the clinical outlook for treatment of hereditary mitochondrial disease.Peer reviewe

Dealing with an Unconventional Genetic Code in Mitochondria: The Biogenesis and Pathogenic Defects of the 5‐Formylcytosine Modification in Mitochondrial tRNAMet^{Met}

Human mitochondria contain their own genome, which uses an unconventional genetic code. In addition to the standard AUG methionine codon, the single mitochondrial tRNA Methionine (mt‐tRNA$^{Met}$) also recognises AUA during translation initiation and elongation. Post‐transcriptional modifications of tRNAs are important for structure, stability, correct folding and aminoacylation as well as decoding. The unique 5‐formylcytosine (f$^{5}$C) modification of position 34 in mt‐tRNA$^{Met}$ has been long postulated to be crucial for decoding of unconventional methionine codons and efficient mitochondrial translation. However, the enzymes responsible for the formation of mitochondrial f$^{5}$C have been identified only recently. The first step of the f$^{5}$C pathway consists of methylation of cytosine by NSUN3. This is followed by further oxidation by ABH1. Here, we review the role of f$^{5}$C, the latest breakthroughs in our understanding of the biogenesis of this unique mitochondrial tRNA modification and its involvement in human disease.Medical Research Council, UK is gratefully acknowledged for generous support of this work

Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin

Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries.

UNLABELLED: Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. IMPORTANCE: Human cytomegalovirus (HCMV), a betaherpesvirus, is a leading cause of morbidity and mortality during congenital infection and among immunosuppressed individuals. HCMV infection significantly changes cellular metabolism. Akin to tumor cells, in HCMV-infected cells, glycolysis is increased and glucose carbon is shifted from the tricarboxylic acid cycle to fatty acid biosynthesis. However, unlike in tumor cells, HCMV induces mitochondrial biogenesis even under aerobic glycolysis. Here, we have affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We find that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.S.K. was supported by a European Molecular Biology Organization long-term fellowship (ALTF 887-2009). M.P.W is funded by a Wellcome Trust Senior Clinical Fellowship (108070/Z/15/Z). R.J.S. is supported by MRC grant (MR/L008734/1). P.J.L . is supported by a Wellcome Trust Principal Research Fellowship, grant (WT101835). J. S. is supported by MRC Programme grant (G0701279). J.R., L. V. and M.M. are supported by MRC as part of the core funding for the Mitochondrial Biology Unit (MC_U105697135). L.V. is also supported by EMBO (ALFT 701- 2013).This is the final version of the article. It first appeared from the American Society for Microbiology via http://dx.doi.org/10.1128/mBio.00029-1

Regulation of Mammalian Mitochondrial Gene Expression: Recent Advances

Perturbation of mitochondrial DNA (mtDNA) gene expression can lead to human pathologies. Therefore, a greater appreciation of the basic mechanisms of mitochondrial gene expression is desirable to understand the pathophysiology of associated disorders. Although the purpose of the mitochondrial gene expression machinery is to provide only 13 proteins of the oxidative phosphorylation (OxPhos) system, recent studies have revealed its remarkable and unexpected complexity. We review here the latest breakthroughs in our understanding of the post-transcriptional processes of mitochondrial gene expression, focusing on advances in analyzing the mitochondrial epitranscriptome, the role of mitochondrial RNA granules (MRGs), the benefits of recently obtained structures of the mitochondrial ribosome, and the coordination of mitochondrial and cytosolic translation to orchestrate the biogenesis of OxPhos complexes