A Forward Genetic Screen Revealed the Role of POLDIP2 in Ensuring Maternal Inheritance of Mitochondrial DNA in Drosophila melanogaster

Abstract

Mitochondria are indispensable organelles for eukaryotes, functioning in energy production and multiple other cellular processes, including calcium homeostasis and apoptosis. They house their own genetic materials known as mitochondrial DNA (mtDNA). In most animals, the inheritance of mtDNA follows a uniparental pattern, generally from the maternal side. Maternal inheritance is accomplished by mechanisms actively degrading paternal mtDNA before and after fertilisation. For many species, including Drosophila, fish and humans, the paternal mitochondrial genomes are eliminated in the late stages of spermatogenesis. In Drosophila melanogaster, two nuclear-encoded mitochondrial proteins, EndoG and PolG1, were shown to participate in the elimination of the paternal mtDNA (DeLuca & O'Farrell, 2012; Yu et al., 2017). However, many details of this process remain uncharacterised. The main objective of this thesis is the identification of other nuclear-encoded proteins and the investigation towards how they regulate the degradation of paternal mtDNA in Drosophila sperm cells. To systematically screen for factors influencing pre-fertilisation mtDNA elimination, I examined 1415 mutant lines carrying random mutations in the nuclear genome (on the 3rd chromosome). This work involved dissecting and staining seminal vesicles with an antibody targeting dsDNA to quantify mtDNA levels in mature sperm. Through the screen, I identified 63 lines that retained mtDNA in their mature sperm. 24 of these lines in addition to 13 lines previously identified by other members of the Ma lab were sequenced, and mutations were identified by bioinformatic pipelines. I then performed deficiency mapping and revealed the involvement of POLDIP2 and three proteins of the procollagen-proline 4-dioxygenase gene group (PH4alphaSG2, CG15539 and CG31524) in the elimination of paternal mtDNA during spermatogenesis. I generated CRISPR mutants and confirmed the role of POLDIP2 in paternal mtDNA elimination. I further demonstrated that overexpression of the POLDIP2-PB isoform in the POLDIP2 mutant background effectively rescued the leakage phenotype, confirming its association with the elimination process. Moreover, I performed genetic crosses and droplet digital PCR and indicated that the retained paternal mtDNA could be transmitted to F1 progeny. Biochemical assays showed that POLDIP2 localises to the mitochondrial matrix and interacts with ClpX, a chaperone-like subunit of the major protease ClpXP in the mitochondria. Mutation of POLDIP2 was previously shown to result in alteration of the cellular levels and activity of ClpXP in human cells, which could affect the maintenance of nucleoids and mtDNA. Indeed, I found that knocking down/out ClpX led to the retention of mtDNA in mature sperm. Therefore, we hypothesise that POLDIP2 and ClpX collaborate to regulate mtDNA dynamics during spermatogenesis. Further experiments are required to test this hypothesis. In conclusion, this thesis identifies multiple factors that ensure the maternal inheritance of mtDNA in Drosophila. Moreover, detailed characterisations of POLDIP2 reveal potential mechanisms on how this protein regulates the elimination of paternal mtDNA in Drosophila sperm cells to ensure maternal inheritance of this genome. This work thus illuminates the evolutionary trajectory of uniparental inheritance and provides insights that could inform strategies for treating pathogenic mtDNA transmission.