The mammalian respiratory chain complexes I, III2, and IV (CI, CIII2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII2-CIV), that is ...biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation.
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•Three amino acids (EED) in UQCRC1 of complex III2 control respirasome stability•Homozygous Uqcrc1delEED knockin mice have drastically reduced respirasome levels•Profound depletion of respirasomes does not impair oxidative phosphorylation•Mice with a profound depletion of respirasomes have apparently normal physiology
The role of the mammalian respirasome, a stable assembly of a complete respiratory chain, is much debated. Milenkovic et al. have generated knockin mice with a profound depletion of respirasomes. Very unexpectedly, the mutant mice have preserved oxidative phosphorylation capacity and physiology, arguing that respirasomes may be dispensable for normal bioenergetics.
Mitochondrial DNA (mtDNA) copy number regulation is altered in several human mtDNA-mutation diseases and it is also important in a variety of normal physiological processes. Mitochondrial ...transcription factor A (TFAM) is essential for human mtDNA transcription and we demonstrate here that it is also a key regulator of mtDNA copy number. We initially performed in vitro transcription studies and determined that the human TFAM protein is a poor activator of mouse mtDNA transcription, despite its high capacity for unspecific DNA binding. Next, we generated P1 artificial chromosome (PAC) transgenic mice ubiquitously expressing human TFAM. The introduced human TFAM gene was regulated in a similar fashion as the endogenous mouse Tfam gene and expression of the human TFAM protein in the mouse did not result in down-regulation of the endogenous expression. The PAC-TFAM mice thus had a net overexpression of TFAM protein and this resulted in a general increase of mtDNA copy number. We used a combination of mice with TFAM overexpression and TFAM knockout and demonstrated that mtDNA copy number is directly proportional to the total TFAM protein levels also in mouse embryos. Interestingly, the expression of human TFAM in the mouse results in up-regulation of mtDNA copy number without increasing respiratory chain capacity or mitochondrial mass. It is thus possible to experimentally dissociate mtDNA copy number regulation from mtDNA expression and mitochondrial biogenesis in mammals in vivo. In conclusion, our results provide genetic evidence for a novel role for TFAM in direct regulation of mtDNA copy number in mammals.
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.
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•MRPP3 is essential and its activity is non-redundant in mitochondria•5′ tRNA cleavage precedes 3′ end processing in vivo•RNA processing is required for biogenesis of mitoribosomes•RNA processing links transcription to translation via mitoribosome assembly
Rackham et al. generate conditional knockout mice lacking the endoribonuclease component of the mitochondrial RNase P and show it is essential. They show that 5′ tRNA cleavage precedes 3′ processing and that processing is required for mitoribosome biogenesis. They find that RNA processing links transcription to translation via mitoribosome assembly.
Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the ...mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.
Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein ...required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase γ. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.
Proteins crucial for the respiratory chain are translated by the mitochondrial ribosome. Mitochondrial ribosome biogenesis is therefore critical for oxidative phosphorylation capacity and ...disturbances are known to cause human disease. This complex process is evolutionary conserved and involves several RNA processing and modification steps required for correct ribosomal RNA maturation. We recently showed that a member of the mitochondrial transcription termination factor (MTERF) family of proteins, MTERF4, recruits NSUN4, a 5-methylcytosine RNA methyltransferase, to the large ribosomal subunit in a process crucial for mitochondrial ribosome biogenesis. Here, we describe the 3D crystal structure of the human MTERF4-NSUN4 complex determined to 2.9 Å resolution. MTERF4 is composed of structurally repeated MTERF-motifs that form a nucleic acid binding domain. NSUN4 lacks an N-or C-terminal extension that is commonly used for RNA recognition by related RNA methyltransferases. Instead, NSUN4 binds to the C-terminus of MTERF4. A positively charged surface forms an RNA binding path from the concave to the convex side of MTERF4 and further along NSUN4 all of the way into the active site. This finding suggests that both subunits of the protein complex likely contribute to RNA recognition. The interface between MTERF4 and NSUN4 contains evolutionarily conserved polar and hydrophobic amino acids, and mutations that change these residues completely disrupt complex formation. This study provides a molecular explanation for MTERF4-dependent recruitment of NSUN4 to ribosomal RNA and suggests a unique mechanism by which other members of the large MTERF-family of proteins can regulate ribosomal biogenesis.
Variants of mitochondrial DNA (mtDNA) are commonly used as markers to track human evolution because of the high sequence divergence and exclusive maternal inheritance. It is assumed that the ...inheritance is clonal, i.e. that mtDNA is transmitted between generations without germline recombination. In contrast to this assumption, a number of studies have reported the presence of recombinant mtDNA molecules in cell lines and animal tissues, including humans. If germline recombination of mtDNA is frequent, it would strongly impact phylogenetic and population studies by altering estimates of coalescent time and branch lengths in phylogenetic trees. Unfortunately, this whole area is controversial and the experimental approaches have been widely criticized as they often depend on polymerase chain reaction (PCR) amplification of mtDNA and/or involve studies of transformed cell lines. In this study, we used an in vivo mouse model that has had germline heteroplasmy for a defined set of mtDNA mutations for more than 50 generations. To assess recombination, we adapted and validated a method based on cloning of single mtDNA molecules in the λ phage, without prior PCR amplification, followed by subsequent mutation analysis. We screened 2922 mtDNA molecules and found no germline recombination after transmission of mtDNA under genetically and evolutionary relevant conditions in mammals.
The 3′ end of the rRNA of the small ribosomal subunit contains two extremely highly conserved dimethylated adenines. This modification and the responsible methyltransferases are present in all three ...domains of life, but its function has remained elusive. We have disrupted the mouse
Tfb1m gene encoding a mitochondrial protein homologous to bacterial dimethyltransferases and demonstrate here that loss of TFB1M is embryonic lethal. Disruption of
Tfb1m in heart leads to complete loss of adenine dimethylation of the rRNA of the small mitochondrial ribosomal subunit, impaired assembly of the mitochondrial ribosome, and abolished mitochondrial translation. In addition, we present biochemical evidence that TFB1M does not activate or repress transcription in the presence of TFB2M. Our results thus show that TFB1M is a nonredundant dimethyltransferase in mammalian mitochondria. In addition, we provide a possible explanation for the universal conservation of adenine dimethylation of rRNA by showing a critical role in ribosome maintenance.
Characterization of the basic transcription machinery of mammalian mitochondrial DNA (mtDNA) is of fundamental biological interest and may also lead to therapeutic interventions for human diseases ...associated with mitochondrial dysfunction. Here we report that mitochondrial transcription factors B1 (TFB1M) and B2 (TFB2M) are necessary for basal transcription of mammalian mitochondrial DNA (mtDNA). Human TFB1M and TFB2M are expressed ubiquitously and can each support promoter-specific mtDNA transcription in a pure recombinant in vitro system containing mitochondrial RNA polymerase (POLRMT) and mitochondrial transcription factor A. Both TFB1M and TFB2M interact directly with POLRMT, but TFB2M is at least one order of magnitude more active in promoting transcription than TFB1M. Both factors are highly homologous to bacterial rRNA dimethyltransferases, which suggests that an RNA-modifying enzyme has been recruited during evolution to function as a mitochondrial transcription factor. The presence of two proteins that interact with mammalian POLRMT may allow flexible regulation of mtDNA gene expression in response to the complex physiological demands of mammalian metabolism.
Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in a variety of tissues during ageing in humans, monkeys and rodents. These mutations are unevenly distributed and can accumulate ...clonally in certain cells, causing a mosaic pattern of respiratory chain deficiency in tissues such as heart, skeletal muscle and brain. In terms of the ageing process, their possible causative effects have been intensely debated because of their low abundance and purely correlative connection with ageing. We have now addressed this question experimentally by creating homozygous knock-in mice that express a proof-reading-deficient version of PolgA, the nucleus-encoded catalytic subunit of mtDNA polymerase. Here we show that the knock-in mice develop an mtDNA mutator phenotype with a threefold to fivefold increase in the levels of point mutations, as well as increased amounts of deleted mtDNA. This increase in somatic mtDNA mutations is associated with reduced lifespan and premature onset of ageing-related phenotypes such as weight loss, reduced subcutaneous fat, alopecia (hair loss), kyphosis (curvature of the spine), osteoporosis, anaemia, reduced fertility and heart enlargement. Our results thus provide a causative link between mtDNA mutations and ageing phenotypes in mammals.
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