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。通过发育的线粒体过程质量的质量为有害线粒体DNA突变的选择性遗传 尽管mtDNA容易突变，并且并非所有常规的DNA修复系统在线粒体中都起作用，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。在这里，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发育的线粒体过程在细胞器级别进行纯化的选择。我们证明了线粒体裂变，加上早期胚芽中缺乏mtDNA复制，实际上将mtDNA分离为单个细胞器。 mtDNA分离后，mtDNA转录开始，导致每个细胞器中的呼吸激活。 mtDNA编码基因的表达允许在异质细胞中不同线粒体基因型的功能表现，因此作为每个单个基因组的应力测试，并为复制竞争奠定了阶段。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。了解在形成卵泡中激活JNK和线粒体生物发生的发育和细胞机制。 卵子发生的是线粒体质量和mtDNA拷贝数的巨大增加，这是提供足够的线粒体供应成熟卵并遏制有害mtDNA变体的传播所必需的。在分裂生殖细胞时，mtDNA复制在果蝇卵巢中的卵母细胞测定时启动，这需要活跃的线粒体呼吸。我们表明，喂食前胰岛素-MYC环路通过增强电子传输链亚基的表达以及MTDNA复制和表达以及线粒体蛋白的进口而促进线粒体呼吸和生物发生。我们进一步揭示了JNK的瞬时激活增强了胰岛素受体的表达，并启动了胰岛素-MYC信号回路。该信号继电器促进了卵巢中的线粒体生物发生，从而在限制有害mtDNA突变的传播中发挥了作用。我们的研究表明，将线粒体生物发生和遗传与卵母细胞发育息息相关的细胞机制。在确定了促进生物发生中的JNK-MYC信号级联，我们对JNK在区分2b胚芽的分化卵泡中的急剧和瞬态激活产生了兴趣。在区域2B中，圆形16细胞囊肿被体细胞封装，该细胞将囊肿压缩到单细胞层盘中。我们的初步研究表明，由体细胞压缩引起的生殖细胞的机械应力激活了伸展的Ca2+通道，这是JNK激活所必需的。现在，我们正在进行遗传分析，以确定可能涉及的细胞内CA2+信号传导中的其他参与者，并描绘整个信号级联反应会导致JNK激活。 项目3。线粒体调节肠道干细胞增殖和上皮稳态通过FOXO 从糖酵解到氧化磷酸化的代谢过渡通常与许多类型的干细胞的分化有关。但是，线粒体呼吸与干细胞行为之间的联系尚不完全了解。我们在果蝇的肠道干细胞（ISC）中遗传破坏了电子传输链（ETC）。我们发现，携带受损等的ISC比正常生产速度慢得多，并且产生的肠细胞很少，因此未能进一步分化为肠细胞。 ISC增殖和谱系规范的主要障碍之一似乎是异常升高的叉子盒O（FoxO）信号在ETC缺乏的ISC中，因为遗传抑制信号通路部分抑制了肠细胞的数量。与共同的信念相反，活性氧（ROS）积累似乎并未介导ET ETC突变表型。我们的结果表明，线粒体呼吸对于果蝇ISC的增殖和体内谱系规范至关重要，并且至少通过抑制内源性FOXO信号传导来部分作用。 项目4。线粒体RNA聚合酶的PPR结构域是mtDNA复制所需的核糖核酸酶 线粒体DNA复制和转录对细胞能代谢至关重要。线粒体RNA聚合酶（POLRRMT）被认为是mtDNA复制的原始酶。然而，尚不清楚通常转录长的多重配合RNA的Polrrmt如何产生短的RNA寡聚来启动mtDNA复制。在这里，我们表明果蝇polrmt的PPR结构域是3至5个驱虫核酸酶。 PPR结构域的驱虫核酸酶活性是必不可少的，即POLRMT在体外合成短RNA寡寡寡寡，并且在体内进行了NOVO MTDNA复制所必需的。成年果蝇中驱虫酶不足的polrmt的过表达导致严重的过早衰老表型和MTDNA转录本错误的中等增加，这表明驱虫核酸酶的活性可能有助于mtDNA转录的校对。同样，人POLRMT中的PPR结构域也具有大核酸酶活性，表明PPR结构域在线粒体DNA和RNA代谢中的进化保守作用。 项目5。研究在Dictyostelium中进口的线粒体tRNA。 对实验室的长期兴趣是开发动物模型中mtDNA转化的方法，该方法将对线粒体基因组进行全面的分析，有助于对人类mtDNA疾病进行建模并促进有效疗法的发展。当前，成功的mtDNA转化是一个主要的技术障碍是缺乏将DNA/RNA引入线粒体活细胞的有效方法。无法使用工程策略将核酸输送到线粒体中，这将我们的注意力转移到了自然上。尽管大多数后生动物线粒体基因组编码了一套RRNA和TRNA的整体，用于翻译mtDNA编码的蛋白质，但许多生物的线粒体基因组缺乏一些或完整的TRNA。他们必须进口在核基因组中编码的TRN，从细胞质到线粒体。因此，我们试图研究原生物迪斯特尔迪斯特尔迪斯特尔的tRNA的分子基础，希望从这项研究中获得的知识将为动物中线粒体转化的未来发展奠定基础。 为了更好地理解在Dicty中进口的线粒体tRNA，我们首先应用定量蛋白质组学，并确定了在Dicty线粒体中高度富集的总共1,061种蛋白质。现在，我们正在使用GFP标记测定法确认其线粒体定位，以验证蛋白质组学数据。我们还正在构建一个合成tRNA阵列以补充内源性tRNA进口系统，这将使我们能够使用补体屏幕识别潜在的tRNA导入机械。