The human mitochondrial genome contains a second light strand promoter.

The human mitochondrial genome must be replicated and expressed in a timely manner to maintain energy metabolism and supply cells with adequate levels of adenosine triphosphate. Central to this process is the idea that replication primers and gene products both arise via transcription from a single light strand promoter (LSP) such that primer formation can influence gene expression, with no consensus as to how this is regulated. Here, we report the discovery of a second light strand promoter (LSP2) in humans, with features characteristic of a bona fide mitochondrial promoter. We propose that the position of LSP2 on the mitochondrial genome allows replication and gene expression to be orchestrated from two distinct sites, which expands our long-held understanding of mitochondrial gene expression in humans.

Dinucleotide Degradation by REXO2 Maintains Promoter Specificity in Mammalian Mitochondria.

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.

Topoisomerase 3α Is Required for Decatenation and Segregation of Human mtDNA.

How mtDNA replication is terminated and the newly formed genomes are separated remain unknown. We here demonstrate that the mitochondrial isoform of topoisomerase 3α (Top3α) fulfills this function, acting independently of its nuclear role as a component of the Holliday junction-resolving BLM-Top3α-RMI1-RMI2 (BTR) complex. Our data indicate that mtDNA replication termination occurs via a hemicatenane formed at the origin of H-strand replication and that Top3α is essential for resolving this structure. Decatenation is a prerequisite for separation of the segregating unit of mtDNA, the nucleoid, within the mitochondrial network. The importance of this process is highlighted in a patient with mitochondrial disease caused by biallelic pathogenic variants in TOP3A, characterized by muscle-restricted mtDNA deletions and chronic progressive external ophthalmoplegia (CPEO) plus syndrome. Our work establishes Top3α as an essential component of the mtDNA replication machinery and as the first component of the mtDNA separation machinery.

Mechanisms and pathologies of human mitochondrial DNA replication and deletion formation.

Human mitochondria possess a multi-copy circular genome, mitochondrial DNA (mtDNA), that is essential for cellular energy metabolism. The number of copies of mtDNA per cell, and their integrity, are maintained by nuclear-encoded mtDNA replication and repair machineries. Aberrant mtDNA replication and mtDNA breakage are believed to cause deletions within mtDNA. The genomic location and breakpoint sequences of these deletions show similar patterns across various inherited and acquired diseases, and are also observed during normal ageing, suggesting a common mechanism of deletion formation. However, an ongoing debate over the mechanism by which mtDNA replicates has made it difficult to develop clear and testable models for how mtDNA rearrangements arise and propagate at a molecular and cellular level. These deletions may impair energy metabolism if present in a high proportion of the mtDNA copies within the cell, and can be seen in primary mitochondrial diseases, either in sporadic cases or caused by autosomal variants in nuclear-encoded mtDNA maintenance genes. These mitochondrial diseases have diverse genetic causes and multiple modes of inheritance, and show notoriously broad clinical heterogeneity with complex tissue specificities, which further makes establishing genotype-phenotype relationships challenging. In this review, we aim to cover our current understanding of how the human mitochondrial genome is replicated, the mechanisms by which mtDNA replication and repair can lead to mtDNA instability in the form of large-scale rearrangements, how rearranged mtDNAs subsequently accumulate within cells, and the pathological consequences when this occurs.

Mammalian RNase H1 directs RNA primer formation for mtDNA replication initiation and is also necessary for mtDNA replication completion.

The in vivo role for RNase H1 in mammalian mitochondria has been much debated. Loss of RNase H1 is embryonic lethal and to further study its role in mtDNA expression we characterized a conditional knockout of Rnaseh1 in mouse heart. We report that RNase H1 is essential for processing of RNA primers to allow site-specific initiation of mtDNA replication. Without RNase H1, the RNA:DNA hybrids at the replication origins are not processed and mtDNA replication is initiated at non-canonical sites and becomes impaired. Importantly, RNase H1 is also needed for replication completion and in its absence linear deleted mtDNA molecules extending between the two origins of mtDNA replication are formed accompanied by mtDNA depletion. The steady-state levels of mitochondrial transcripts follow the levels of mtDNA, and RNA processing is not altered in the absence of RNase H1. Finally, we report the first patient with a homozygous pathogenic mutation in the hybrid-binding domain of RNase H1 causing impaired mtDNA replication. In contrast to catalytically inactive variants of RNase H1, this mutant version has enhanced enzyme activity but shows impaired primer formation. This finding shows that the RNase H1 activity must be strictly controlled to allow proper regulation of mtDNA replication.

Two type I topoisomerases maintain DNA topology in human mitochondria.

Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.

Separating and Segregating the Human Mitochondrial Genome.

Cells contain thousands of copies of the mitochondrial genome. These genomes are distributed within the tubular mitochondrial network, which is itself spread across the cytosol of the cell. Mitochondrial DNA (mtDNA) replication occurs throughout the cell cycle and ensures that cells maintain a sufficient number of mtDNA copies. At replication termination the genomes must be resolved and segregated within the mitochondrial network. Defects in mtDNA replication and segregation are a cause of human mitochondrial disease associated with failure of cellular energy production. This review focuses upon recent developments on how mitochondrial genomes are physically separated at the end of DNA replication, and how these genomes are subsequently segregated and distributed around the mitochondrial network.

Understanding mitochondrial DNA maintenance disorders at the single muscle fibre level.

Clonal expansion of mitochondrial DNA (mtDNA) deletions is an important pathological mechanism in adults with mtDNA maintenance disorders, leading to a mosaic mitochondrial respiratory chain deficiency in skeletal muscle. This study had two aims: (i) to determine if different Mendelian mtDNA maintenance disorders showed similar pattern of mtDNA deletions and respiratory chain deficiency and (ii) to investigate the correlation between the mitochondrial genetic defect and corresponding respiratory chain deficiency. We performed a quantitative analysis of respiratory chain deficiency, at a single cell level, in a cohort of patients with mutations in mtDNA maintenance genes. Using the same tissue section, we performed laser microdissection and single cell genetic analysis to investigate the relationship between mtDNA deletion characteristics and the respiratory chain deficiency. The pattern of respiratory chain deficiency is similar with different genetic defects. We demonstrate a clear correlation between the level of mtDNA deletion and extent of respiratory chain deficiency within a single cell. Long-range and single molecule PCR shows the presence of multiple mtDNA deletions in approximately one-third of all muscle fibres. We did not detect evidence of a replicative advantage for smaller mtDNA molecules in the majority of fibres, but further analysis is needed to provide conclusive evidence.

MGME1 processes flaps into ligatable nicks in concert with DNA polymerase γ during mtDNA replication.

Recently, MGME1 was identified as a mitochondrial DNA nuclease with preference for single-stranded DNA (ssDNA) substrates. Loss-of-function mutations in patients lead to mitochondrial disease with DNA depletion, deletions, duplications and rearrangements. Here, we assess the biochemical role of MGME1 in the processing of flap intermediates during mitochondrial DNA replication using reconstituted systems. We show that MGME1 can cleave flaps to enable efficient ligation of newly replicated DNA strands in combination with POLγ. MGME1 generates a pool of imprecisely cut products (short flaps, nicks and gaps) that are converted to ligatable nicks by POLγ through extension or excision of the 3'-end strand. This is dependent on the 3'-5' exonuclease activity of POLγ which limits strand displacement activity and enables POLγ to back up to the nick by 3'-5' degradation. We also demonstrate that POLγ-driven strand displacement is sufficient to generate DNA- but not RNA-flap substrates suitable for MGME1 cleavage and ligation during replication. Our findings have implications for RNA primer removal models, the 5'-end processing of nascent DNA at OriH, and DNA repair.

Mutations in GTPBP3 cause a mitochondrial translation defect associated with hypertrophic cardiomyopathy, lactic acidosis, and encephalopathy.

Respiratory chain deficiencies exhibit a wide variety of clinical phenotypes resulting from defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mtDNA or mutations in nuclear genes coding for mitochondrial proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial physiology. By whole-exome and candidate gene sequencing, we identified 11 individuals from 9 families carrying compound heterozygous or homozygous mutations in GTPBP3, encoding the mitochondrial GTP-binding protein 3. Affected individuals from eight out of nine families presented with combined respiratory chain complex deficiencies in skeletal muscle. Mutations in GTPBP3 are associated with a severe mitochondrial translation defect, consistent with the predicted function of the protein in catalyzing the formation of 5-taurinomethyluridine (τm(5)U) in the anticodon wobble position of five mitochondrial tRNAs. All case subjects presented with lactic acidosis and nine developed hypertrophic cardiomyopathy. In contrast to individuals with mutations in MTO1, the protein product of which is predicted to participate in the generation of the same modification, most individuals with GTPBP3 mutations developed neurological symptoms and MRI involvement of thalamus, putamen, and brainstem resembling Leigh syndrome. Our study of a mitochondrial translation disorder points toward the importance of posttranscriptional modification of mitochondrial tRNAs for proper mitochondrial function.

Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease.

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA.

ELAC2 mutations cause a mitochondrial RNA processing defect associated with hypertrophic cardiomyopathy.

The human mitochondrial genome encodes RNA components of its own translational machinery to produce the 13 mitochondrial-encoded subunits of the respiratory chain. Nuclear-encoded gene products are essential for all processes within the organelle, including RNA processing. Transcription of the mitochondrial genome generates large polycistronic transcripts punctuated by the 22 mitochondrial (mt) tRNAs that are conventionally cleaved by the RNase P-complex and the RNase Z activity of ELAC2 at 5' and 3' ends, respectively. We report the identification of mutations in ELAC2 in five individuals with infantile hypertrophic cardiomyopathy and complex I deficiency. We observed accumulated mtRNA precursors in affected individuals muscle and fibroblasts. Although mature mt-tRNA, mt-mRNA, and mt-rRNA levels were not decreased in fibroblasts, the processing defect was associated with impaired mitochondrial translation. Complementation experiments in mutant cell lines restored RNA processing and a yeast model provided additional evidence for the disease-causal role of defective ELAC2, thereby linking mtRNA processing to human disease.

Mitochondrial transcript maturation and its disorders.

Mitochondrial respiratory chain deficiencies exhibit a wide spectrum of clinical presentations owing to defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mitochondrial DNA (mtDNA) or mutations in nuclear genes coding for mitochondrially-targeted proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial biology including expression of mtDNA-encoded genes. Expression of the mitochondrial genes is extensively regulated at the post-transcriptional stage and entails nucleolytic cleavage of precursor RNAs, RNA nucleotide modifications, RNA polyadenylation, RNA quality and stability control. These processes ensure proper mitochondrial RNA (mtRNA) function, and are regulated by dedicated, nuclear-encoded enzymes. Recent growing evidence suggests that mutations in these nuclear genes, leading to incorrect maturation of RNAs, are a cause of human mitochondrial disease. Additionally, mutations in mtDNA-encoded genes may also affect RNA maturation and are frequently associated with human disease. We review the current knowledge on a subset of nuclear-encoded genes coding for proteins involved in mitochondrial RNA maturation, for which genetic variants impacting upon mitochondrial pathophysiology have been reported. Also, primary pathological mtDNA mutations with recognised effects upon RNA processing are described.

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.

Isolating Mitochondria, Mitoplasts, and mtDNA from Cultured Mammalian Cells.

Mitochondria are double membrane-bound eukaryotic organelles with roles in a range of cellular activities including energy conversion, apoptosis, cell signalling, and the biosynthesis of enzyme cofactors. Mitochondria contain their own genome, called mtDNA, which encodes subunits of the oxidative phosphorylation machinery as well as the rRNA and tRNA molecules required for their translation within mitochondria. The ability to isolate highly purified mitochondria from cells has been instrumental in a number of studies of mitochondrial function. Differential centrifugation is a long-established method for the isolation of mitochondria. Cells are subjected to osmotic swelling and disruption, followed by centrifugation in isotonic sucrose solutions to separate mitochondria from other cellular components. We present a method using this principle for the isolation of mitochondria from cultured mammalian cell lines. Mitochondria purified by this method can be further fractionated to investigate protein localization, or act as a starting point to purify mtDNA.

Pathological variants in TOP3A cause distinct disorders of mitochondrial and nuclear genome stability.

Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.

Controlling the topology of mammalian mitochondrial DNA.

The genome of mitochondria, called mtDNA, is a small circular DNA molecule present at thousands of copies per human cell. MtDNA is packaged into nucleoprotein complexes called nucleoids, and the density of mtDNA packaging affects mitochondrial gene expression. Genetic processes such as transcription, DNA replication and DNA packaging alter DNA topology, and these topological problems are solved by a family of enzymes called topoisomerases. Within mitochondria, topoisomerases are involved firstly in the regulation of mtDNA supercoiling and secondly in disentangling interlinked mtDNA molecules following mtDNA replication. The loss of mitochondrial topoisomerase activity leads to defects in mitochondrial function, and variants in the dual-localized type IA topoisomerase TOP3A have also been reported to cause human mitochondrial disease. We review the current knowledge on processes that alter mtDNA topology, how mtDNA topology is modulated by the action of topoisomerases, and the consequences of altered mtDNA topology for mitochondrial function and human health.

The mitochondrial single-stranded DNA binding protein is essential for initiation of mtDNA replication.

We report a role for the mitochondrial single-stranded DNA binding protein (mtSSB) in regulating mitochondrial DNA (mtDNA) replication initiation in mammalian mitochondria. Transcription from the light-strand promoter (LSP) is required both for gene expression and for generating the RNA primers needed for initiation of mtDNA synthesis. In the absence of mtSSB, transcription from LSP is strongly up-regulated, but no replication primers are formed. Using deep sequencing in a mouse knockout model and biochemical reconstitution experiments with pure proteins, we find that mtSSB is necessary to restrict transcription initiation to optimize RNA primer formation at both origins of mtDNA replication. Last, we show that human pathological versions of mtSSB causing severe mitochondrial disease cannot efficiently support primer formation and initiation of mtDNA replication.

The Maintenance of Mitochondrial DNA Integrity and Dynamics by Mitochondrial Membranes.

Mitochondria are complex organelles that harbour their own genome. Mitochondrial DNA (mtDNA) exists in the form of a circular double-stranded DNA molecule that must be replicated, segregated and distributed around the mitochondrial network. Human cells typically possess between a few hundred and several thousand copies of the mitochondrial genome, located within the mitochondrial matrix in close association with the cristae ultrastructure. The organisation of mtDNA around the mitochondrial network requires mitochondria to be dynamic and undergo both fission and fusion events in coordination with the modulation of cristae architecture. The dysregulation of these processes has profound effects upon mtDNA replication, manifesting as a loss of mtDNA integrity and copy number, and upon the subsequent distribution of mtDNA around the mitochondrial network. Mutations within genes involved in mitochondrial dynamics or cristae modulation cause a wide range of neurological disorders frequently associated with defects in mtDNA maintenance. This review aims to provide an understanding of the biological mechanisms that link mitochondrial dynamics and mtDNA integrity, as well as examine the interplay that occurs between mtDNA, mitochondrial dynamics and cristae structure.

Recurrent horizontal transfer identifies mitochondrial positive selection in a transmissible cancer.

Autonomous replication and segregation of mitochondrial DNA (mtDNA) creates the potential for evolutionary conflict driven by emergence of haplotypes under positive selection for 'selfish' traits, such as replicative advantage. However, few cases of this phenomenon arising within natural populations have been described. Here, we survey the frequency of mtDNA horizontal transfer within the canine transmissible venereal tumour (CTVT), a contagious cancer clone that occasionally acquires mtDNA from its hosts. Remarkably, one canine mtDNA haplotype, A1d1a, has repeatedly and recently colonised CTVT cells, recurrently replacing incumbent CTVT haplotypes. An A1d1a control region polymorphism predicted to influence transcription is fixed in the products of an A1d1a recombination event and occurs somatically on other CTVT mtDNA backgrounds. We present a model whereby 'selfish' positive selection acting on a regulatory variant drives repeated fixation of A1d1a within CTVT cells.