Mapping and editing animal mitochondrial genomes: can we overcome the challenges?

The animal mitochondrial genome, although small, can have a big impact on health and disease. Non-pathogenic sequence variation among mitochondrial DNA (mtDNA) haplotypes influences traits including fertility, healthspan and lifespan, whereas pathogenic mutations are linked to incurable mitochondrial diseases and other complex conditions like ageing, diabetes, cancer and neurodegeneration. However, we know very little about how mtDNA genetic variation contributes to phenotypic differences. Infrequent recombination, the multicopy nature and nucleic acid-impenetrable membranes present significant challenges that hamper our ability to precisely map mtDNA variants responsible for traits, and to genetically modify mtDNA so that we can isolate specific mutants and characterize their biochemical and physiological consequences. Here, we summarize the past struggles and efforts in developing systems to map and edit mtDNA. We also assess the future of performing forward and reverse genetic studies on animal mitochondrial genomes. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.This work is funded by Wellcome Trust grant 203767/Z/16/Z to AK and 202269/Z/16/Z to HM

A battle for transmission: the cooperative and selfish animal mitochondrial genomes.

The mitochondrial genome is an evolutionarily persistent and cooperative component of metazoan cells that contributes to energy production and many other cellular processes. Despite sharing the same host as the nuclear genome, the multi-copy mitochondrial DNA (mtDNA) follows very different rules of replication and transmission, which translate into differences in the patterns of selection. On one hand, mtDNA is dependent on the host for its transmission, so selections would favour genomes that boost organismal fitness. On the other hand, genetic heterogeneity within an individual allows different mitochondrial genomes to compete for transmission. This intra-organismal competition could select for the best replicator, which does not necessarily give the fittest organisms, resulting in mito-nuclear conflict. In this review, we discuss the recent advances in our understanding of the mechanisms and opposing forces governing mtDNA transmission and selection in bilaterians, and what the implications of these are for mtDNA evolution and mitochondrial replacement therapy

Two mitochondrial DNA polymorphisms modulate cardiolipin binding and lead to synthetic lethality

Genetic screens have been used extensively to probe interactions betweennuclear genes and their impact on phenotypes. Probing interactions betweenmitochondrial genes and their phenotypic outcome, however, has not beenpossible due to a lack of tools to map the responsible polymorphisms. Here,using a toolkit we previously established in Drosophila, we isolate over 300recombinant mitochondrial genomes and map a naturally occurring polymorphism at the cytochrome c oxidase III residue 109 (CoIII109) that fully rescues the lethality and other defects associated with a point mutation incytochrome c oxidase I (CoIT300I). Through lipidomics profiling, biochemicalassays and phenotypic analyses, we show that the CoIII109 polymorphismmodulates cardiolipin binding to prevent complex IV instability caused by theCoIT300I mutation. This study demonstrates the feasibility of genetic interactionscreens in animal mitochondrial DNA. It unwraps the complex intra-genomicinterplays underlying disorders linked to mitochondrial DNA and how theyinfluence disease expression

Identification of the critical replication targets of CDK reveals direct regulation of replication initiation factors by the embryo polarity machinery in C. elegans

During metazoan development, the cell cycle is remodelled to coordinate proliferation with differentiation. Developmental cues cause dramatic changes in the number and timing of replication initiation events, but the mechanisms and physiological importance of such changes are poorly understood. Cyclin-dependent kinases (CDKs) are important for regulating S-phase length in many metazoa, and here we show in the nematode Caenorhabditis elegans that an essential function of CDKs during early embryogenesis is to regulate the interactions between three replication initiation factors SLD-3, SLD-2 and MUS-101 (Dpb11/TopBP1). Mutations that bypass the requirement for CDKs to generate interactions between these factors is partly sufficient for viability in the absence of Cyclin E, demonstrating that this is a critical embryonic function of this Cyclin. Both SLD-2 and SLD-3 are asymmetrically localised in the early embryo and the levels of these proteins inversely correlate with S-phase length. We also show that SLD-2 asymmetry is determined by direct interaction with the polarity protein PKC-3. This study explains an essential function of CDKs for replication initiation in a metazoan and provides the first direct molecular mechanism through which polarization of the embryo is coordinated with DNA replication initiation factors

Identification of the critical replication targets of CDK reveals direct regulation of replication initiation factors by the embryo polarity machinery in C. elegans

During metazoan development, the cell cycle is remodelled to coordinate proliferation with differentiation. Developmental cues cause dramatic changes in the number and timing of replication initiation events, but the mechanisms and physiological importance of such changes are poorly understood. Cyclin-dependent kinases (CDKs) are important for regulating S-phase length in many metazoa, and here we show in the nematode Caenorhabditis elegans that an essential function of CDKs during early embryogenesis is to regulate the interactions between three replication initiation factors SLD-3, SLD-2 and MUS-101 (Dpb11/TopBP1). Mutations that bypass the requirement for CDKs to generate interactions between these factors is partly sufficient for viability in the absence of Cyclin E, demonstrating that this is a critical embryonic function of this Cyclin. Both SLD-2 and SLD-3 are asymmetrically localised in the early embryo and the levels of these proteins inversely correlate with S-phase length. We also show that SLD-2 asymmetry is determined by direct interaction with the polarity protein PKC-3. This study explains an essential function of CDKs for replication initiation in a metazoan and provides the first direct molecular mechanism through which polarization of the embryo is coordinated with DNA replication initiation factors

How to Mend a Broken Mitochondrial Genome: Mitochondrial Recombination And Its Applications

Animal mitochondria produce cellular energy and rely on genes encoded within their own genome (mtDNA) in addition to those in the nucleus. mtDNA mutations are linked to mitochondrial and age-related diseases. Therefore, safeguarding mechanisms are crucial to limit mtDNA mutations and curtail ageing and disease. Yet, pathways that repair animal mtDNA double-strand breaks remain uncharacterised. Double-strand breaks can be accurately repaired by homologous recombination but spontaneous mtDNA recombination is only detected rarely in animals. A system to induce and select for mtDNA recombination was recently developed in the fruit fly, Drosophila melanogaster. In this thesis, I used this system to investigate the mechanism of animal mtDNA recombination and its applications for mitochondrial genetics. First, I identified candidates that may mediate mtDNA recombination by performing a screen for nuclear repair proteins that are enriched in mitochondria when overexpressed in Drosophila cell culture. This identified mitochondrial localisation of REC, a helicase that drives meiotic crossovers in the nucleus. I then performed mtDNA recombination assays and found that REC is required for mtDNA recombination in Drosophila germline and somatic tissues. Moreover, loss of REC increased age-induced mitochondrial mutation load and dysfunction, showing that REC safeguards mtDNA during ageing. Next, I investigated whether the mitochondrial function of REC is conserved in humans. The human homologue of REC, MCM8, and its interacting partner, MCM9, localise to mitochondria and human cells with mutated MCM8 accumulate more mtDNA mutations. Therefore, MCM8 also functions to safeguard mtDNA. To gain further insight into the mechanism of Drosophila mtDNA recombination, I examined whether other nuclear recombination factors localise to mitochondria in vivo. Nearly all factors examined were not enriched in mitochondria, suggesting that mitochondrial recombination may occur by a different mechanism to that in the nucleus. Finally, I utilised the system to induce mtDNA recombination to develop forward and reverse genetic tools to study mtDNA. Techniques to edit animal mtDNA are desperately needed to better understand the consequences of mtDNA mutations on disease and ageing

REC drives recombination to repair double-strand breaks in animal mtDNA.

Funder: Gurdon Institute Core FacilityMechanisms that safeguard mitochondrial DNA (mtDNA) limit the accumulation of mutations linked to mitochondrial and age-related diseases. Yet, pathways that repair double-strand breaks (DSBs) in animal mitochondria are poorly understood. By performing a candidate screen for mtDNA repair proteins, we identify that REC-an MCM helicase that drives meiotic recombination in the nucleus-also localizes to mitochondria in Drosophila. We show that REC repairs mtDNA DSBs by homologous recombination in somatic and germline tissues. Moreover, REC prevents age-associated mtDNA mutations. We further show that MCM8, the human ortholog of REC, also localizes to mitochondria and limits the accumulation of mtDNA mutations. This study provides mechanistic insight into animal mtDNA recombination and demonstrates its importance in safeguarding mtDNA during ageing and evolution.ERC starting gran

Two mitochondrial DNA polymorphisms modulate cardiolipin binding and lead to synthetic lethality

Acknowledgements: We thank Professor Frank Jiggins from the University of Cambridge for providing the ten D. melanogaster stocks collected from the wild in the UK (Fig. 3c). We also would like to acknowledge the Gurdon Institute Core Facilities for their general support and the Metabolomics Core Facility at the Institute of Physiology of the Czech Academy of Sciences for conducting LC-MS-based lipidomics profiling. This work is funded by ERC Starting Grant 803852, Wellcome Trust Sir Henry Dale Fellowship 202269/Z/16/B, Philip Leverhulme Prize PLP-2020-063 and an EMBO Small Grant to H.M. The Gurdon Institute Core Facility is funded by Wellcome Trust grant 203144 and Cancer Research UK grant C6946/A24843. The Laboratory of Mitochondrial Physiology, Institute of Physiology of the Czech Academy of Sciences, is funded by the Grant Agency of the Czech Republic grants 22-17173S and 21-01205S.Funder: European Molecular Biology Organization (EMBO); doi: https://doi.org/10.13039/100004410AbstractGenetic screens have been used extensively to probe interactions between nuclear genes and their impact on phenotypes. Probing interactions between mitochondrial genes and their phenotypic outcome, however, has not been possible due to a lack of tools to map the responsible polymorphisms. Here, using a toolkit we previously established in Drosophila, we isolate over 300 recombinant mitochondrial genomes and map a naturally occurring polymorphism at the cytochrome c oxidase III residue 109 (CoIII109) that fully rescues the lethality and other defects associated with a point mutation in cytochrome c oxidase I (CoIT300I). Through lipidomics profiling, biochemical assays and phenotypic analyses, we show that the CoIII109 polymorphism modulates cardiolipin binding to prevent complex IV instability caused by the CoIT300I mutation. This study demonstrates the feasibility of genetic interaction screens in animal mitochondrial DNA. It unwraps the complex intra-genomic interplays underlying disorders linked to mitochondrial DNA and how they influence disease expression.</jats:p