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Mitochondrial DNA genetics inheritance

线粒体DNA遗传学遗传

基本信息

批准号：
10003781
负责人：
Hong Xu
金额：
$ 236.7万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/10003781
关键词：
5&apos -exoribonuclease Adenosine Triphosphate Adult Amoeba genus Animal Model Animals Bacterial Proteins Balbiani Body Behavior Biogenesis Bioinformatics Breathing Cell Differentiation process Cell Extracts Cell Maintenance Cell Proliferation Cells Complex Couples DNA Repair DNA biosynthesis DNA-Directed RNA Polymerase Defect Development Dictyostelium discoideum Drosophila genus Electron Transport Electroporation Energy Metabolism Ensure Enterocytes Enteroendocrine Cell Exoribonucleases Female Funding Future Genetic Genetic Transcription Genome Genotype Germ Cells Glutamic Acid Glycolysis Goals Growth Homeostasis Human Impairment Individual Insulin Intestines Ions Life Link MAPK8 gene Masks Mass Spectrum Analysis Methods Minor Mitochondria Mitochondrial DNA Mitochondrial Inheritance Mitochondrial Matrix Mitochondrial Proteins Mitochondrial RNA Modeling Molecular Molecular Analysis Mothers Mutation Nucleic Acids Oocytes Oogenesis Organelles Organism Outer Mitochondrial Membrane Ovary Oxidative Phosphorylation PINK1 gene Pathway interactions Phenotype Point Mutation Population Premature aging syndrome Preparation Procedures Process Production Proliferating Proline Protein Biosynthesis Protein Import Protein Kinase Proteins Proteome Proteomics RNA RNA primers Reactive Oxygen Species Regulation Research Respiration Rest Ribonucleases Role Secure Series Signal Pathway Signal Transduction Stem cells Stress Tests System Techniques Testing Transcript Transfection Transfer RNA Translating Translations Transplantation Variant Work base divalent metal egg fitness fly genetic analysis genetic manipulation human disease in vivo insulin signaling mitochondrial DNA mutation mitochondrial genome notch protein novel nucleocytoplasmic transport offspring prevent progenitor protein expression repaired segregation social stem stem cell differentiation tool transmission process

项目摘要

Project 1. Mitochondrial behaviors prime the selective inheritance against harmful mitochondrial DNA mutations Although mitochondrial DNA is prone to mutation and few mtDNA repair mechanisms exist, deleterious mutations are exceedingly rare. How the transmission of detrimental mtDNA mutations are restricted through the maternal lineage is debated. 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 behaviors. We demonstrate that mitochondrial fission, together with the lack of mtDNA replication in proliferating germ cells, effectively segregates mtDNA into individual organelles. After mtDNA segregation, mtDNA expression begins, which leads to the activation of respiration in each organelle. The expression of mtDNA 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. Electron transport chain biogenesis activated by a JNK-insulin-Myc relay primes mitochondrial inheritance in Drosophila Mitochondrial contents and activities are tightly controlled according to the cellular energy demand and specific developmental regulations. Oogenesis features an enormous increase in mitochondrial mass and mtDNA copy number to furnish mature egg and prime a competitive replication to curb the transmission of deleterious mtDNA variants. Nonetheless, it is unclear how the massive mitochondrial biogenesis and mtDNA replication are triggered and maintained during oogenesis. Here, we demonstrate an insulin-Myc signaling loop that boosts the expression of essential factors for mtDNA replication and expression, energy metabolism, and protein import in the Drosophila ovary. We also reveal that a transient activation of JNK activity is required to initiate the Myc-insulin signaling loop. Importantly, this signaling relay ensures sufficient mitochondrial contents in eggs and limits the transmission of a deleterious mtDNA variant. This work demonstrates a developmental regulation that couples oocyte growth with mtDNA proliferation and selective inheritance. Project 3. PINK1 Inhibits Local Protein Synthesis to Limit Transmission of Deleterious Mitochondrial DNA Mutations We have previously proposed that selective inheritance, the limited transmission of damaging mtDNA mutations from mother to offspring, is based on replication competition in Drosophila. This model, which stems from our observation that wild-type mitochondria propagate much more vigorously in the fly ovary than mitochondria carrying fitness-impairing mutations, implies that germ cells recognize the fitness of individual mitochondria, and selectively boost the propagation of healthy ones. Here, we demonstrate that the protein kinase PINK1 preferentially accumulates on mitochondria enriched for a deleterious mtDNA mutation. PINK1 phosphorylates Larp to inhibit protein synthesis on the mitochondrial outer membrane. Impaired local translation on defective mitochondria in turn limits the replication of their mtDNA, and hence the transmission of deleterious mutations to the offspring. Our work confirms that selective inheritance occurs at the organelle level during Drosophila oogenesis, and provides molecular entry points to test this model in other systems. Project 4. Mitochondrial OXPHOS regulates Drosophila intestinal stem cells differentiation through FOXO and Notch pathways. Stem cells often rely on glycolysis for energy production, and switching to mitochondrial oxidative phosphorylation (OXPHOS) is believed to be essential for stem cell differentiation. However, the link between mitochondrial OXPHOS and stem cell differentiation remains to be explored. We tackled this question by genetically disrupting mitochondrial OXPHOS in the intestinal stem cells (ISCs) of Drosophila. We found that ISCs carrying dysfunctional mitochondria divided much more slowly than normal and produced very few intestinal progenitors, or enteroblasts (EBs), which themselves failed to differentiate into enterocytes (ECs) or enteroendocrine cells (EEs). Further studies revealed abnormaly elevated FOXO and Notch signaling in the OXPHOS-defective ISCs, which may be the main impediment to ISCs differentiation into ECs and EEs, as genetically suppressing the two signaling pathways partially rescues the differentiation defect. Our results demonstrate that mitochondrial OXPHOS is essential for Drosophila ISC proliferation and differentiation in vivo, and acts at least partially repressing endogenous FOXO and Notch signaling. Project 5. Pentatricopeptide repeats of mitochondrial RNA polymerase is an exoribonuclease and required for DNA replication and transcription proofreading We identified that the pentatricopeptide repeat (PPR) domain in mitochondrial RNA polymerase (mtRNApol) possesses RNase activity and is essential for primer synthesis of mtDNA replication. Bacterial protein expression of PPR domain hydrolyzes RNA substrates in a 3-5 manner and requires divalent metal ions for its activity. We further showed that a point mutation of glutamic acid to proline in PPR domain (E423P) causes loss of RNase activity. The E423P mutation fails to synthesize RNA primers for mtDNA replication but retains the RNA polymerase function. We also demonstrated flies over-expressing E423P in adult stage had significantly increased incorporation errors in mitochondrial transcripts, and demonstrated many premature aging phenotypes. In additional, the RNase activity of PPR domain in mtRNApol is highly conserved between Drosophila and human. Our work defines a novel function for PPR domain as a 3-5 exoribonuclease and its conserved roles in in mtDNA replication and transcription proofreading. Project 6 Characterizing the mitochondrial proteome of Dictyostelium discoideum using quantitative mass spectroscopy Currently, there is no method to transform mitochondria in animal cells. The major hurdle toward a successful mitochondrial transformation is to effectively deliver nucleic acids into the mitochondrial matrix. Curiously, mitochondrial transformation was successfully achieved in Dictyostelium discoideum using the routine electroporation procedure, suggesting Dicty mitochondria are naturally competent. Consistent with this notion Dicty does not possess a full suite of mitochondrial tRNAs on mtDNA, and must transport nuclear-encoded tRNAs into the mitochondria to translate mtDNA-encoded proteins, underscoring the presence of nucleic acids importing machinery on Dicty mitochondria. To better understand the mitochondria tRNA importing process, we applied quantitative proteomic approaches, to characterize mitochondrial proteome in Dicty. We recovered 1,200 proteins from the purified Dicty mitochondria and were enriched in the highly purified mitochondrial preparation compared total cell extracts. Bioinformatic analyses revealed that about 200 Dicty specific mitochondrial proteins constitute candidates for future genetic analysis to identify factors required for tRNA import.This work is part of a larger study to characterize the mechanism in D. discoideum responsible for nucleic acid import into the mitochondria with a long-term aspiration of transplanting a minimal system of mitochondrial nucleic acid import into other model organisms and enabling mitochondrial transfection in animal cells.

项目1。线粒体行为为选择性遗传对抗有害的线粒体DNA突变提供了基础