Mitochondrial DNA genetics inheritance

线粒体DNA遗传学遗传

• 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
• 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' -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

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. 线粒体行为引发针对有害线粒体 DNA 突变的选择性遗传 尽管线粒体DNA很容易发生突变并且线粒体DNA修复机制很少存在，但有害突变却极其罕见。如何限制有害 mtDNA 突变通过母系的传播仍存在争议。我们利用果蝇来剖析 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发育协调的线粒体行为在细胞器水平上进行纯化选择。我们证明线粒体裂变以及增殖生殖细胞中线粒体DNA复制的缺乏，有效地将线粒体DNA分离到单个细胞器中。 mtDNA 分离后，mtDNA 开始表达，从而激活每个细胞器的呼吸作用。线粒体DNA的表达允许异质细胞中不同线粒体基因型的功能表现，因此可以作为每个个体基因组的压力测试，并为复制竞争奠定基础。我们还表明，巴尔比亚尼体通过向极质提供健康的线粒体，在线粒体 DNA 选择性遗传中发挥次要作用。这两种选择机制可能协同作用，以确保功能性线粒体DNA通过果蝇卵子发生的传递。 项目 2. JNK-胰岛素-Myc 中继激活的电子传递链生物发生启动果蝇线粒体遗传 线粒体的含量和活动根据细胞能量需求和特定的发育规律受到严格控制。卵子发生的特点是线粒体质量和 mtDNA 拷贝数大幅增加，以提供成熟的卵子并引发竞争性复制，以遏制有害 mtDNA 变异的传播。尽管如此，目前尚不清楚卵子发生过程中大量线粒体生物发生和线粒体DNA复制是如何触发和维持的。在这里，我们展示了一个胰岛素-Myc 信号环路，它可以增强果蝇卵巢中 mtDNA 复制和表达、能量代谢和蛋白质输入的必需因子的表达。我们还揭示了 JNK 活性的瞬时激活是启动 Myc-胰岛素信号环路所必需的。重要的是，这种信号传递确保鸡蛋中有足够的线粒体含量，并限制有害线粒体 DNA 变异的传播。这项工作展示了一种将卵母细胞生长与 mtDNA 增殖和选择性遗传结合起来的发育调控。 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 我们之前提出选择性遗传，即破坏性线粒体DNA突变从母亲到后代的有限传播，是基于果蝇的复制竞争。该模型源于我们的观察，即野生型线粒体在果蝇卵巢中比携带健康受损突变的线粒体繁殖得更加活跃，这意味着生殖细胞识别单个线粒体的健康度，并选择性地促进健康线粒体的繁殖。在这里，我们证明蛋白激酶 PINK1 优先在富含有害 mtDNA 突变的线粒体上积累。 PINK1 磷酸化 Larp 以抑制线粒体外膜上的蛋白质合成。有缺陷的线粒体上的局部翻译受损反过来又限制了线粒体DNA的复制，从而限制了有害突变向后代的传播。我们的工作证实了果蝇卵子发生过程中细胞器水平上发生选择性遗传，并提供了在其他系统中测试该模型的分子切入点。 项目 4. 线粒体 OXPHOS 通过 FOXO 和 Notch 途径调节果蝇肠干细胞分化。 干细胞通常依靠糖酵解来产生能量，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。然而，线粒体 OXPHOS 和干细胞分化之间的联系仍有待探索。我们通过基因破坏果蝇肠干细胞 (ISC) 中的线粒体 OXPHOS 解决了这个问题。我们发现携带功能障碍线粒体的ISC分裂速度比正常细胞慢得多，并且产生很少的肠祖细胞或肠成细胞（EB），它们本身无法分化为肠上皮细胞（EC）或肠内分泌细胞（EE）。进一步的研究表明，OXPHOS 缺陷的 ISC 中 FOXO 和 Notch 信号通路异常升高，这可能是 ISC 分化为 EC 和 EE 的主要障碍，因为从基因上抑制这两种信号通路可以部分挽救分化缺陷。我们的结果表明，线粒体 OXPHOS 对于果蝇 ISC 体内增殖和分化至关重要，并且至少部分抑制内源性 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一种核糖核酸外切酶，是 DNA 复制和转录校对所必需的 我们发现线粒体 RNA 聚合酶 (mtRNApol) 中的五肽重复 (PPR) 结构域具有 RNase 活性，并且对于 mtDNA 复制的引物合成至关重要。 PPR 结构域的细菌蛋白表达以 3-5 方式水解 RNA 底物，并需要二价金属离子才能发挥其活性。我们进一步表明，PPR 结构域 (E423P) 中谷氨酸点突变为脯氨酸会导致 RNase 活性丧失。 E423P突变无法合成用于mtDNA复制的RNA引物，但保留了RNA聚合酶功能。我们还证明，在成年阶段过度表达 E423P 的果蝇会显着增加线粒体转录本中的掺入错误，并表现出许多过早衰老的表型。此外，mtRNApol 中 PPR 结构域的 RNase 活性在果蝇和人类之间高度保守。我们的工作将 PPR 结构域的新功能定义为 3-5 核糖核酸外切酶，及其在 mtDNA 复制和转录校对中的保守作用。 项目 6 使用定量质谱表征盘基网柄菌线粒体蛋白质组 目前，还没有方法可以转化动物细胞中的线粒体。成功线粒体转化的主要障碍是有效地将核酸递送到线粒体基质中。奇怪的是，使用常规电穿孔程序在盘基网柄菌中成功实现了线粒体转化，这表明盘基网柄菌线粒体具有天然能力。与这一观点相一致的是，Dicty 并不拥有 mtDNA 上的全套线粒体 tRNA，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，这强调了 Dicty 线粒体上存在核酸输入机制。为了更好地了解线粒体 tRNA 导入过程，我们应用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，并与总细胞提取物相比，在高度纯化的线粒体制剂中富集。生物信息学分析显示，大约 200 种 Dicty 特异性线粒体蛋白构成了未来遗传分析的候选蛋白，以鉴定 tRNA 输入所需的因子。这项工作是一项更大研究的一部分，该研究旨在描述 D. discoideum 中负责核酸输入线粒体的机制，其中长期愿望是将最小的线粒体核酸输入系统移植到其他模型生物体中，并在动物细胞中实现线粒体转染。