Mitochondrial DNA genetics inheritance

线粒体DNA遗传学遗传

• 批准号: 10253873
• 负责人: Hong Xu
• 金额: $ 150.47万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10253873
• 关键词: 5' -exoribonuclease; Adenosine Triphosphate; Adult; Amoeba genus; Animal Model; Animals; Attention; Balbiani Body; Belief; Biogenesis; Biological Assay; Breathing; Cell Differentiation process; Cell Lineage; Cell Maintenance; Cell membrane; Cell physiology; Cells; Complement; Complex; Cyst; Cytoplasm; DNA; DNA Primase; DNA Repair; DNA biosynthesis; DNA metabolism; DNA-Directed RNA Polymerase; Data; Development; Dictyostelium; Dictyostelium discoideum; Disease; Drosophila genus; Electron Transport; Encapsulated; Energy Metabolism; Engineering; Enterocytes; Epithelial; Epithelium; Exoribonucleases; Female; Foundations; Funding; Future; Genes; Genetic; Genetic Transcription; Genetic study; Genome; Genotype; Germ Cells; Glycolysis; Goals; Homeostasis; Human; Impairment; In Vitro; Individual; Insulin; Insulin Receptor; Intestines; Knowledge; Life; Link; MAPK8 gene; Masks; Mechanical Stress; Mediating; Metabolic; Methods; Minor; Mitochondria; Mitochondrial DNA; Mitochondrial Matrix; Mitochondrial Proteins; Mitochondrial RNA; Modeling; Molecular; Molecular Analysis; Mutation; Nature; Nuclear; Nucleic Acids; Oligonucleotides; Oocytes; Oogenesis; Operating System; Organelles; Organism; Ovary; Oxidative Phosphorylation; PINK1 gene; Pancreatic ribonuclease; Phenotype; Phosphotransferases; Play; Population; Premature aging syndrome; Process; Proliferating; Proteins; Proteomics; RNA; RNA metabolism; Reactive Oxygen Species; Regulation; Research; Respiration; Rest; Ribosomal RNA; Role; Secure; Series; Signal Pathway; Signal Transduction; Somatic Cell; Stress Tests; Stretching; System; Techniques; Testing; Transcript; Transfer RNA; Translations; Variant; Work; base; cell behavior; effective therapy; egg; fitness; fly; genetic analysis; human disease; human model; in vivo; interest; mitochondrial DNA mutation; mitochondrial genome; mutant; overexpression; prevent; segregation; social; stem cell differentiation; stem cell proliferation; stem cells; tool; transmission process

Project 1. Developmentally-orchestrated mitochondrial processes prime the selective inheritance against harmful mitochondrial DNA mutations Although mtDNA is prone to mutation and not all conventional DNA repair systems operate in mitochondria, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations is restricted through the maternal lineage is debated. Here, we use Drosophila to dissect the mechanisms of mtDNA selective inheritance and understand their molecular underpinnings. Our observations support a purifying selection at the organelle level based on a series of developmentally-orchestrated mitochondrial processes. We demonstrate that mitochondrial fission, together with the lack of mtDNA replication in early germarium, effectively segregates mtDNA into individual organelles. After mtDNA segregation, mtDNA transcription begins, which leads to the activation of respiration in each organelle. The expression of mtDNA-encoded genes allows the functional manifestation of different mitochondrial genotypes in heteroplasmic cells, and hence functions as a stress test for each individual genome and sets the stage for the replication competition. We also show that the Balbiani body has a minor role in mtDNA selective inheritance by supplying healthy mitochondria to the pole plasm. The two selection mechanisms may act synergistically to secure the transmission of functional mtDNA through Drosophila oogenesis. Project 2. Understand the developmental and cellular mechanisms activating JNK and mitochondrial biogenesis in forming follicle. Oogenesis features an enormous increase in mitochondrial mass and mtDNA copy number, which are required to furnish mature eggs with an adequate supply of mitochondria and to curb the transmission of deleterious mtDNA variants. Quiescent in dividing germ cells, mtDNA replication initiates upon oocyte determination in the Drosophila ovary, which necessitates active mitochondrial respiration. We showed that an feedforward insulin-Myc loop promotes mitochondrial respiration and biogenesis by boosting the expression of electron transport chain subunits and of factors essential for mtDNA replication and expression, and for the import of mitochondrial proteins. We further revealed that transient activation of JNK enhances the expression of the insulin receptor and initiates the insulin-Myc signaling loop. This signaling relay promotes mitochondrial biogenesis in the ovary, and thereby plays a role in limiting the transmission of deleterious mtDNA mutations. Our study demonstrates cellular mechanisms that couple mitochondrial biogenesis and inheritance with oocyte development. Having identified a JNK-Myc signaling cascade in promoting ETC biogenesis, we are intrigued by the sharp and transient activation of JNK in differentiating follicles at region 2B germarium. In region 2B, the round 16-cell cyst is encapsulated by somatic cells, which compress the cyst into a single-cell layer disc. Our preliminary studies suggest that mechanical stress on germ cells plasma membrane caused by somatic cells compression activates stretch-sensitive Ca2+ channel, which is required for JNK activation. We are now carrying out genetic analyses to identify additional players in intracellular Ca2+ signaling that might be involved, and to delineate the whole signaling cascade leads to the JNK activation. Project 3. Mitochondria regulate intestinal stem cell proliferation and epithelial homeostasis through FOXO A metabolic transition from glycolysis to oxidative phosphorylation is often associated with differentiation of many types of stem cells. However, the link between mitochondrial respiration and stem cells behavior is not fully understood. We genetically disrupted electron transport chain (ETC) complexes in the intestinal stem cells (ISCs) of Drosophila. We found that ISCs carrying impaired ETC proliferated much more slowly than normal and produced very few enteroblasts, which failed to further differentiate into enterocytes. One of the main impediments to ISC proliferation and lineage specification appeared to be abnormally elevated forkhead box O (FOXO) signaling in the ETC-deficient ISCs, as genetically suppressing the signaling pathway partially restored the number of enterocytes. Contrary to common belief, reactive oxygen species (ROS) accumulation did not appear to mediate the ETC mutant phenotype. Our results demonstrate that mitochondrial respiration is essential for Drosophila ISC proliferation and lineage specification in vivo and acts at least partially by repressing endogenous FOXO signaling. Project 4. The PPR domain of mitochondrial RNA polymerase is a ribonuclease required for mtDNA replication Mitochondrial DNA 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 of PPR domain is indispensable for POLRMT to synthesize short RNA oligos in vitro and required for de novo mtDNA replication in vivo. Overexpression of exoribonuclease deficient POLRMT in adult flies leads to severe premature aging phenotypes and a moderate increase of mtDNA transcripts errors, suggesting that exoribonuclease activity may contribute to the proofreading of mtDNA transcription. Similarly, PPR domain in human POLRMT also has exoribonuclease activity, indicating evolutionarily conserved roles of PPR domain in mitochondrial DNA and RNA metabolism. Project 5. Study mitochondrial tRNA importing in Dictyostelium. A long-term interest in the lab is to develop methods for mtDNA transformation in animal models, which would enable comprehensive analyses of mitochondrial genome, help to model human mtDNA diseases, and to facilitate the development of effective therapies. Currently, a major technical hurdle toward a successful mtDNA transformation is the lack of an effective way to introduce DNA/RNA into mitochondria of living cells. The inability to deliver nucleic acids into mitochondria using engineering strategies has shifted our attention to nature. While most metazoans mitochondrial genomes encode a full suite of rRNAs and tRNAs for the translation of mtDNA encoded proteins, many protists mitochondrial genomes lack a few or the complete set of tRNAs. They have to import tRNAs that are encoded in the nuclear genome, from cytoplasm to mitochondria. We hence seek to study the molecular underpins of tRNA importing in a protist, Dictyostelium discoideum, hoping that the knowledge gained from this study will lay the foundation for future development on mitochondrial transformation in animals. To better understand mitochondrial tRNA importing in Dicty, we first applied quantitative proteomics and identified total 1,061 proteins that were highly enriched in Dicty mitochondria. We are now using GFP tagging assay to confirm their mitochondrial localization, to validate the proteomic data. We are also constructing a synthetic tRNA array to complement endogenous tRNA importing system, which will allow us to identify potential tRNA importing machinery using complementation screen.

项目1。发育协调的线粒体过程启动了选择性遗传，以对抗有害的线粒体DNA突变