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

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

Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large‐scale deletions or point mutations

We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site‐specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the “common deletion” (CD), a 4977‐bp repeat‐flanked deletion associated with adult‐onset chronic progressive external ophthalmoplegia and, less frequently, Kearns‐Sayre and Pearson's marrow pancreas syndromes. Expression of mtZFNs led to a reduction in mutant mtDNA haplotype load, and subsequent repopulation of wild‐type mtDNA restored mitochondrial respiratory function in a CD cybrid cell model. This study constitutes proof‐of‐principle that, through heteroplasmy manipulation, delivery of site‐specific nuclease activity to mitochondria can alleviate a severe biochemical phenotype in primary mitochondrial disease arising from deleted mtDNA species

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

PDE12 removes mitochondrial RNA poly(A) tails and controls translation in human mitochondria

Polyadenylation of mRNA in human mitochondria is crucial for gene expression and perturbation of poly(A) tail length has been linked to a human neurodegenerative disease. Here we show that 2′-phosphodiesterase (2′-PDE), (hereafter PDE12), is a mitochondrial protein that specifically removes poly(A) extensions from mitochondrial mRNAs both in vitro and in mitochondria of cultured cells. In eukaryotes, poly(A) tails generally stabilize mature mRNAs, whereas in bacteria they increase mRNA turnover. In human mitochondria, the effects of increased PDE12 expression were transcript dependent. An excess of PDE12 led to an increase in the level of three mt-mRNAs (ND1, ND2 and CytB) and two (CO1 and CO2) were less abundant than in mitochondria of control cells and there was no appreciable effect on the steady-state level of the remainder of the mitochondrial transcripts. The alterations in poly(A) tail length accompanying elevated PDE12 expression were associated with severe inhibition of mitochondrial protein synthesis, and consequently respiratory incompetence. Therefore, we propose that mRNA poly(A) tails are important in regulating protein synthesis in human mitochondria, as it is the case for nuclear-encoded eukaryotic mRNA

Myosin VI-Dependent Actin Cages Encapsulate Parkin-Positive Damaged Mitochondria.

Mitochondrial quality control is essential to maintain cellular homeostasis and is achieved by removing damaged, ubiquitinated mitochondria via Parkin-mediated mitophagy. Here, we demonstrate that MYO6 (myosin VI), a unique myosin that moves toward the minus end of actin filaments, forms a complex with Parkin and is selectively recruited to damaged mitochondria via its ubiquitin-binding domain. This myosin motor initiates the assembly of F-actin cages to encapsulate damaged mitochondria by forming a physical barrier that prevents refusion with neighboring populations. Loss of MYO6 results in an accumulation of mitophagosomes and an increase in mitochondrial mass. In addition, we observe downstream mitochondrial dysfunction manifesting as reduced respiratory capacity and decreased ability to rely on oxidative phosphorylation for energy production. Our work uncovers a crucial step in mitochondrial quality control: the formation of MYO6-dependent actin cages that ensure isolation of damaged mitochondria from the network

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.Medical Research Council, UK; EMBO Fellowship [ALTF 701-2013 to L.V.H.]; PhD fellowship from the Foundation for Science and Technology, Portugal through the GABBA Program, University of Porto (to P.R.G.); Experiments undertaken in the J-PC laboratory were supported by ANR Investissement d’Avenir [ANR-IIINSB-0014] and AFM [18566].This is the final version of the article. It first appeared from Oxford University Press via http://dx.doi.org/10.1093/nar/gkw67

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

A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes