Mitochondrial DNA genetics inheritance

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Project 1. Candidate screening for transcriptional regulations controlling mitochondrial biogenesis Mitochondrial biogenesis is critical to maintain cellular energy homeostasis. In principle, the energetic demand of a cell and the capacity of mitochondria to carry out this need may determine the total mitochondrial mass. However, little is known about mechanisms controlling the basal level of mitochondrial biogenesis, i.e., what are the cellular processes determining the steady-state mitochondrial content in a cell? how do cells sense the mitochondrial abundance and respond accordingly? We have taken advantage of previously developed mitochondrial genetics mosaic system to create mtDNA deficiency in developing eye, which allowed us to carry modifier genetic screen for nuclear factors controlling mitochondrial biogenesis. From the primary RNAi screen over 700 nuclear transcription factors or transcription regulators, we recovered total 23 enhancers and 5 suppressors, which seemingly promote or inhibit mitochondrial biogenesis, respectively. We now performing secondary assays to further validate their roles in mitochondrial biogenesis by directly assessing mitochondria mass, morphology and positioning (SDHB-GFP), and mtDNA contents (TFAM-GFP). Our ongoing study has identified a previously unnoted Zinc finger protein, encoded by CG1663 locus, was essential for the maintenance of both mitochondrial mass and mtDNA. Null mutant of CG1663 is lethal, died at the first instar larva stage. Tissue specific knock down of CG1663 greatly diminished mitochondrial genome, nuclear encoded factors required for mtDNA maintenance and expression, as well as the overall mitochondrial mass. CG1633 protein is highly conserved in metazoan. We are now carrying out ChipSeq and RNAseq to identify the targets of CG1633, aiming to elucidate the potentially conserved transcriptional network controlling mitochondrial biogenesis in animals. Project 2. Mechano-signaling activates oxidative phosphorylation through a Ca2+-CaMK-I cascade in developing oocytes A metabolic transition from glycolysis to oxidative phosphorylation (OXPHOS) is an essential step during stem cell differentiation and tissue maturation. While OXPHOS is favored by unicellular organisms when starved, the transition to OXPHOS is defined in multicellular lives. The mechanisms that induce OXPHOS during differentiation are still unknown. Here, we use the Drosophila ovarian cyst differentiation as an in vivo model and show that altered cell mechanics induces OXPHOS along cell differentiation. After being encased by somatic cells, differentiating cysts are compressed, leading to expansion of the plasma membrane. Membrane expansion promotes Ca2+ influx into the cytosol through Tmc, a stretch-activated ion channel (SAC). Sufficient cytosolic Ca2+ activates OXPHOS and mtDNA replication in differentiating cysts through a signaling cascade. The mechanotransduction pathway is required to support female fertility. Further studies show that SACs mediate contraction-induced OXPHOS during maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our study demonstrates that mechanical activation of mitochondrial energy metabolism is evolutionarily conserved among animals, with implications for association between altered cell mechanics and metabolic disorders such as infertility, cardiovascular diseases, and cancer. Project 3. The PPR domain of mitochondrial RNA polymerase is a ribonuclease required for mtDNA replication Mitochondrial DNA (mtDNA) replication and transcription are of paramount importance to cellular energy metabolism. Mitochondrial RNA polymerase (POLRMT) is thought to be the primase for mtDNA replication. However, it is unclear how POLRMT, which normally transcribes long polycistronic RNAs, can produce short RNA oligos to initiate mtDNA replication. Here we show that the PPR domain of Drosophila POLRMT is a 3 to 5 exoribonuclease. The exoribonuclease activity is indispensable for POLRMT to synthesize short RNA oligos and to prime DNA replication in vitro. An exoribonuclease deficient POLRMT, POLRMTE423P partially restores mitochondrial transcription but fails to support mtDNA replication when expressed in POLRMT mutant background, indicating that the exoribonuclease activity is necessary for mtDNA replication. Overexpression of POLRMTE423P in adult flies leads to severe neuromuscular defects and a marked increase of mtDNA transcripts errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription. PPR domain of human POLRMT also has exoribonuclease activity, indicating evolutionarily conserved roles of PPR domain in mitochondrial DNA and RNA metabolism. Project 4: PolDIP2 is a novel mitochondrial DNA exonuclease essential for eliminating paternal mitochondrial genomes Mitochondrial genome is usually inherited from mother in animals. The uniparental inheritance was regarded as the passive outcome of the distinct cytoplasmic contents of eggs and sperms. Recent studies suggested that active mechanisms also exist to remove the mitochondrial DNA (mtDNA) during spermatogenesis. However, why the paternal mtDNA needs to be eliminated and the protein factors essential for this process are not clear. In this project, we found an under characterized protein called PolDIP2 (DNA Polymerase Delta Interacting Protein 2) is a mtDNA exonuclease that is essential for removing mtDNA in mature sperms in Drosophila. PolDIP2 protein is specifically enriched in Drosophila testis, particularly, in late spermatogenesis stage, when the mitochondrial genome disappears abruptly. In vitro assay suggests PolDIP2 is an exonuclease that digest both single-stranded DNA and double-stranded DNA with sticky ends. PolDIP2 knockout Drosophila is overall healthy but male semi-sterile. In knockout fly, the mtDNA cannot be effectively eliminated in the mature sperms, leaving free-end genomes in the mitochondria; further digesting those remaining mtDNA by ectopically expressing a mitochondrial targeted E.coli exonuclease (mito-Exonuclease III) in sperms can rescue the fertility. Surprisingly, there is a significant elevation of nuclear DNA fragmentation in mature sperms in knockout flies. These results imply that persisting paternal mtDNA could cause deleterious effect on the sperm fitness, likely through the crosstalk between mitochondrial and nuclear DNA regulation. PolDIP2 could be a major factor preventing the transmission of paternal mtDNA to the offspring, and safeguarding the detrimental consequence caused by the persisting mtDNA. Project 6. Construction of a comprehensive compendium of mitochondrial proteins in Dictyostelium discoideum Dictyostelium discoideum, a social amoeba, has emerged as an attractive model to study mitochondrial biology and mitochondrial regulation of cell differentiation and multicellular development. However, a comprehensive list of the mitochondrial proteins has yet to be established in Dictyostelium. We utilized high-throughput multiplexed protein quantitation and homology analyses to generate a high-confidence mitochondrial protein compendium, consisting of 1058 proteins, of which 604 had not been previously annotated. Our proteomic approach, which utilizes a mitochondrial relative enrichment ratio, was validated through mitochondrial targeting sequence (MTS) prediction and live-cell imaging. Our D. discoideum mitochondrial proteome reveals numerous proteins involved in the process of gene expression that are not present in humans, yeasts, or ancestral alphaproteobacteria, which can serve as a foundation for future investigations into the unique mitochondrial biology in D. discoideum. Additionally, we examined the essentiality of respiratory chain complexes for Dictyostelium development.