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

Maturation of selected human mitochondrial tRNAs requires deadenylation

Human mitochondria contain a genome (mtDNA) that encodes essential subunits of the oxidative phosphorylation system. Expression of mtDNA entails multi-step maturation of precursor RNA. In other systems, the RNA life cycle involves surveillance mechanisms, however, the details of RNA quality control have not been extensively characterised in human mitochondria. Using a mitochondrial ribosome profiling and mitochondrial poly(A)-tail RNA sequencing (MPAT-Seq) assay, we identify the poly(A)-specific exoribonuclease PDE12 as a major factor for the quality control of mitochondrial non-coding RNAs. The lack of PDE12 results in a spurious polyadenylation of the 3' ends of the mitochondrial (mt-) rRNA and mt-tRNA. While the aberrant adenylation of 16S mt-rRNA did not affect the integrity of the mitoribosome, spurious poly(A) additions to mt-tRNA led to reduced levels of aminoacylated pool of certain mt-tRNAs and mitoribosome stalling at the corresponding codons. Therefore, our data uncover a new, deadenylation-dependent mtRNA maturation pathway in human mitochondria.This work has been supported by the core funding provided by the Medical Research Council, UK (MC_U105697135). PR-G is supported by Fundação para a Ciência e a Tecnologia, Portugal (PD/BD/105750/2014). AEF is funded by a grant from the Wellcome Trust, UK (106207)

Linear mitochondrial DNA is rapidly degraded by components of the replication machinery.

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5'-3'exonuclease MGME1, elimination of the 3'-5'exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1

The Pseudouridine Synthase RPUSD4 Is an Essential Component of Mitochondrial RNA Granules

Mitochondrial gene expression is a fundamental process that is largely dependent on nuclear-encoded proteins. Several steps of mitochondrial RNA processing and maturation, including RNA post-transcriptional modification, appear to be spatially organized into distinct foci, which we have previously termed mitochondrial RNA granules (MRGs). Although an increasing number of proteins have been localized to MRGs, a comprehensive analysis of the proteome of these structures is still lacking. Here, we have applied a microscopy-based approach that has allowed us to identify novel components of the MRG proteome. Among these, we have focused our attention on RPUSD4, an uncharacterized mitochondrial putative pseudouridine synthase. We show that RPUSD4 depletion leads to a severe reduction of the steady-state level of the 16S mitochondrial (mt) rRNA with defects in the biogenesis of the mitoribosome large subunit and consequently in mitochondrial translation. We report that RPUSD4 binds 16S mt-rRNA, mt-tRNA(Met), and mt-tRNA(Phe), and we demonstrate that it is responsible for pseudouridylation of the latter. These data provide new insights into the relevance of RNA pseudouridylation in mitochondrial gene expression.This work was supported by the Swiss National Science Foundation Grant SNF310030B_160257/1, Wellcome Trust Grant 096919/Z/11/Z, and core funding of Medical Research Council, UK

Mutations in ELAC2 associated with hypertrophic cardiomyopathy impair mitochondrial tRNA 3'-end processing

Mutations in either the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA metabolism, including ELAC2. The ELAC2 gene codes for the mitochondrial RNase Z, responsible for endonucleolytic cleavage of the 3' ends of mitochondrial pre-tRNAs. Here, we report the identification of sixteen novel ELAC2 variants in individuals presenting with mitochondrial respiratory chain deficiency, hypertrophic cardiomyopathy and lactic acidosis. We provide evidence for the pathogenicity of the novel missense variants by studying the RNase Z activity in an in vitro system. We also modelled the residues affected by missense mutation in solved RNase Z structures, providing insight into enzyme structure and function. Finally, we show that primary fibroblasts from the affected individuals have elevated levels of unprocessed mitochondrial RNA precursors. Our study thus broadly confirms the correlation of ELAC2 variants with severe infantile-onset forms of hypertrophic cardiomyopathy and mitochondrial respiratory chain dysfunction. One rare missense variant associated with the occurrence of prostate cancer (p.Arg781His) impairs the mitochondrial RNase Z activity of ELAC2, suggesting a functional link between tumorigenesis and mitochondrial RNA metabolism

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

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp(-/-) mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp(-/-) MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia.This study was supported by the German BMBF and Horizon2020 through E-Rare project GENOMIT (01GM1603 and 01GM1207 to H.P.; FWF-I 2741-B26 to J.A.M.); Vereinigung zur Förderung Pädiatrischer Forschung Salzburg; EU FP7 MEET Project (317433 to H.P. and J.A.M.); Horizon2020 Project SOUND (633974 to H.P.); Marie Skłodowska-Curie Actions Reintegration Fellowship (Mitobiopath-705560 to C.G.); UK NHS Highly Specialised Mitochondrial Service (R.W.T.); Wellcome Centre for Mitochondrial Research (203105/Z/16 to Z.M.C.-L., R.N.L., and R.W.T.); MRC Centre for Neuromuscular Diseases (G0601943 to R.W.T. and P.F.C.); Lily Foundation (R.W.T. and K.T.); UK NIHR fellowship (NIHR-HCS-D12-03-04 to C.L.A.); Wellcome Senior Fellowship (101876/Z/13/Z to P.F.C.); UK NIHR award and MRC Mitochondrial Biology Unit (MC_UP_1501/2 to P.F.C.); NIH (R01 GM0077465 and R35 GM122455 to V.K.M.); EMBO fellowship (ALTF 554-2015 to A.A.J.); UK MRC core funding for the Mitochondrial Biology Unit of the University of Cambridge (MC_U105697135 to A.R.D., P.R.G., and M. Minczuk); Portuguese Fundação para a Ciência e a Tecnologia (PD/BD/105750/2014 to P.R.G.); Italian Telethon (GSP16001 to G.P.C.); Fondazione Cariplo (2014-1010 to D.R.); Strategic Research Center in Private Universities from MEXT; and Practical Research Project for Rare/Intractable Diseases from AMED

Biallelic C1QBP Mutations Cause Severe Neonatal-, Childhood-, or Later-Onset Cardiomyopathy Associated with Combined Respiratory-Chain Deficiencies

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals’ samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp−/− mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp−/− MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia

Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs

Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases