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

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

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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。线粒体行为质量的选择性遗传针对有害线粒体DNA突变尽管线粒体DNA容易突变，并且很少有mtDNA修复机制，但有害突变极为罕见。辩论如何通过母体血统限制有害mtDNA突变的传播。我们使用果蝇来剖析mtDNA选择性遗传的机制，并了解其分子基础。我们的观察结果支持基于一系列具有发育的线粒体行为的细胞器级别纯化选择。我们证明了线粒体裂变，以及在增殖细胞增殖细胞中缺乏mtDNA复制，有效地将mtDNA分离到单个细胞器中。 mtDNA分离后，mtDNA表达开始，从而导致每个细胞器中的呼吸激活。 mtDNA的表达允许在异质细胞中不同线粒体基因型的功能表现，因此可以作为每个单个基因组的应力测试，并为复制竞争奠定了基础。我们还表明，Balbiani体在MTDNA选择性遗传中具有较小的作用，通过向极点质等离子体提供健康的线粒体。两种选择机制可以协同起作用，以确保功能性mtDNA通过果蝇的传播。项目2。通过JNK-胰岛素Myc接力素的电子传输链生物发生在果蝇中的线粒体遗传根据细胞能量需求和特定的发育法规，线粒体内容和活动受到严格控制。卵子发生的是线粒体质量和mtDNA拷贝数的巨大增加，以提供成熟的鸡蛋和竞争性复制，以遏制有害mtDNA变体的传播。但是，目前尚不清楚如何在卵子发生过程中触发和维持大量的线粒体生物发生和mtDNA复制。在这里，我们展示了一种胰岛素-MYC信号环路，该环路可以增强MTDNA复制和表达，能量代谢以及在果蝇卵巢中进口的蛋白质的基本因素的表达。我们还揭示了JNK活性的瞬态激活以启动MYC-胰岛素信号循环。重要的是，该信号继电器可确保卵中足够的线粒体含量，并限制了有害mtDNA变体的传播。这项工作表明了一种发展调节，即卵母细胞生长与mtDNA增殖和选择性遗传。项目3。PINK1抑制局部蛋白质合成以限制有害线粒体DNA突变的传播我们先前曾提出，选择性继承，即有限的损害MTDNA突变从母亲到后代的传播有限，是基于果蝇的复制竞争。该模型源于我们观察到的，即野生型线粒体在卵巢中比携带健身障碍突变的线粒体更剧烈地传播，这意味着生殖细胞识别单个线粒体的适应性，并有选择地增强了健康的线粒体的繁殖。在这里，我们证明蛋白激酶PINK1优先积聚在富含有害mtDNA突变的线粒体上。 Pink1磷酸化LARP以抑制线粒体外膜上的蛋白质合成。线粒体有缺陷的局部翻译反过来限制了其mtDNA的复制，从而将有害突变传播到后代。我们的工作证实，选择性遗传发生在果蝇期间的细胞器水平上，并提供了分子入口点以在其他系统中测试该模型。项目4。线粒体Oxphos通过Foxo和Notch途径调节果蝇干细胞分化。干细胞通常依赖于糖酵解来产生能量，而切换到线粒体氧化磷酸化（OXPHOS）被认为对于干细胞分化至关重要。但是，线粒体oxphos和干细胞分化之间的联系仍有待探索。我们通过遗传破坏果蝇肠道干细胞（ISC）中的线粒体oxphos来解决这个问题。我们发现，携带功能障碍线粒体的ISC比正常分裂得多，很少产生肠道祖细胞或肠细胞（EBS），而肠子本身未能分化为肠上皮细胞（ECS）或肠内分泌细胞（EES）。进一步的研究表明，OXPHOS缺陷ISC中FOXO和Notch信号的升高，这可能是ISC分化为ECS和EES的主要障碍，因为基因抑制了两种信号通路的部分抑制了分化缺陷。我们的结果表明，线粒体oxphos对于果蝇ISC的增殖和体内分化至关重要，并且至少可以部分抑制内源性FOXO和Notch信号传导。项目5。线粒体RNA聚合酶的五肽重复序列是驱精酶，是DNA复制和转录校对所必需的我们确定线粒体RNA聚合酶（MTRNAPOL）中的五肽重复（PPR）结构域具有RNase活性，对于MTDNA复制的启动合成至关重要。 PPR结构域的细菌蛋白表达以3-5的方式水解RNA底物，并且需要二价金属离子才能进行活性。我们进一步表明，谷氨酸在PPR结构域（E423p）中向脯氨酸的点突变会导致RNase活性的丧失。 E423P突变未能合成MTDNA复制的RNA引物，但保留了RNA聚合酶功能。我们还证明了成人阶段中过度表达的E423p的苍蝇在线粒体转录物中的掺入误差显着增加，并证明了许多早熟表型。另外，在果蝇和人之间，PPR结构域的RNase活性高度保守。我们的工作将PPR结构域的新功能定义为3-5驱核核酸酶及其在mtDNA复制和转录校对中的保守作用。项目6使用定量质谱法表征了dictyostelium discoideum的线粒体蛋白质组当前，没有方法可以转化动物细胞中的线粒体。成功的线粒体转化的主要障碍是有效地将核酸输送到线粒体基质中。奇怪的是，使用常规电穿孔程序成功地在迪斯特尔迪斯特尔（Dictyostelium Discoideum）中成功实现了线粒体转化，这表明dicty线粒体自然具有胜任。与这个概念一致，dicty在mtDNA上不具有完整的线粒体TRNA套件，并且必须将核编码的TRNA转运到线粒体中以翻译mtDNA编码的蛋白质，从而强调在Dicty Mitochonchria上导入核酸的核酸的存在。为了更好地理解线粒体tRNA进口过程，我们采用了定量蛋白质组学方法来表征dicty中的线粒体蛋白质组。我们从纯化的dicty线粒体中回收了1,200种蛋白质，并在高度纯化的线粒体制备中富集了总细胞提取物。 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动物细胞中的线粒体转染。