Biochemistry in situ to determine inheritance of RNA-protein complexes

原位生物化学测定 RNA-蛋白质复合物的遗传

• 批准号: 10274800
• 负责人: Scott T Aoki
• 金额: $ 38.99万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-24 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10274800
• 关键词: Affect; Animal Model; Animals; Biochemistry; Caenorhabditis elegans; Cell Culture Techniques; Cells; Cytoplasmic Granules; DNA Modification Process; Development; Enzymes; Family; Foundations; Future Generations; Gene Expression Regulation; Generations; Genes; Germ Cells; Goals; Histones; Immunity; In Situ; Inherited; Label; Ligands; Methods; Molecular; Molecular Biology; Mothers; Mus; Nematoda; Nutrient; Organism; Parents; Physiologic pulse; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Testing; Time; Tissues; Work; chromatin remodeling; imaging capabilities; in vivo; insight; interest; non-genetic; novel; offspring; organizational structure; protein complex; tumor

Biochemistry in situ to determine inheritance of RNA-protein complexes The long-term goal is to elucidate the molecular mechanisms how macromolecular RNA-protein complexes transmit information to future generations of cells and progeny. We classically think of DNA and DNA modifications as the only information inherited between cells. Recent work demonstrates that RNA and RNA- binding proteins are also inherited and that these proteins have functions in organism development and immunity. My lab aims to identify which RNA binding proteins are inherited, determine the macromolecular organization of inherited RNA-protein complexes, and discover the molecular purpose of inheriting these protein complexes. This interest in inherited RNA binding proteins currently extends to investigating RNA-protein complexes that form multi-component RNA-protein granules, or biomolecular condensates. Much of my previous work centered on characterizing the structural organization of P granules, an inherited RNA-protein granule necessary for C. elegans nematode germ cell development. The next five years will focus on understanding the molecular mechanisms how RNA-protein complexes are inherited across cell generations and from parent to progeny. Which RNA binding proteins are inherited, and which cells inherit these proteins? What protein attributes are required for inheritance? Our ability to investigate these questions is currently limited by available methods to track protein components in multicellular organisms. My lab seeks to label and follow maternal proteins in the authentic germline tissue of C. elegans, a proven model organism to study basic questions in animal development. Established single gene editing methods, robust imaging capabilities, and short generational time make C. elegans an ideal multicellular animal to identify the functions of maternal proteins inherited across generations. Modified enzyme tags now allow us to pulse label proteins with covalently bound ligands and chase these labeled proteins over time. This in vivo pulse-chase method has been used to follow chromatin remodeling in cell culture and protein stability in mouse tumors. Our preliminary results demonstrate that we are able to use in vivo pulse chase to track histone protein stability in worm germline tissue under different nutrient conditions. The current goal is to use in vivo pulse chase in C. elegans to visualize the stability of maternal germline RNA binding proteins through germ cell development and track these proteins as they are inherited from mother to progeny. First, we will pulse-label a P granule assembly protein and test how granule formation and protein quantity affect its inheritance. Second, we will pulse-chase maternal germline Argonautes, a family of RNA regulatory enzymes, to identify which Argonautes are inherited by progeny, what tissues inherit them, and what protein attributes are necessary for tissue-specific inheritance. Collectively, this work will redefine our concept of maternal inheritance and elucidate criteria for the inheritance of specific RNA binding proteins. In vivo pulse-chase in C. elegans will provide a foundation to discover novel maternally inherited proteins associated with gene regulation for development, immunity, and beyond. 1

用生物化学原位检测RNA-蛋白质复合体的遗传 长期目标是阐明大分子rna-蛋白质复合体的分子机制。 将信息传递给后代细胞和后代。我们经典地认为DNA和DNA 修改是细胞之间继承的唯一信息。最近的研究表明，RNA和RNA- 结合蛋白也是遗传的，这些蛋白在生物体发育和 豁免权。我的实验室的目标是确定哪些RNA结合蛋白是遗传的，确定大分子 组织遗传的RNA-蛋白质复合体，并发现遗传这些蛋白质的分子目的 复合体。这种对遗传的RNA结合蛋白的兴趣目前扩展到对RNA-蛋白质的研究 形成多组分的RNA-蛋白质颗粒或生物分子凝聚体的复合体。我以前的大部分时间 集中研究P颗粒的结构组织，P颗粒是一种遗传的RNA-蛋白质颗粒 线虫生殖细胞发育所必需的。未来五年将重点了解 RNA-蛋白质复合体如何跨细胞世代和从亲本遗传到亲本的分子机制 后代。哪些RNA结合蛋白是遗传的，哪些细胞继承了这些蛋白质？什么蛋白质 继承需要属性吗？我们调查这些问题的能力目前受到可用资源的限制 追踪多细胞生物体中蛋白质成分的方法。我的实验室试图给孕妇贴上标签并跟踪 线虫正宗生殖系组织中的蛋白质，已被证明是研究基本问题的模式生物 动物发育。成熟的单基因编辑方法、强大的成像能力和简短的 世代时间使线虫成为鉴定母体蛋白功能的理想多细胞动物 世代相传。修改后的酶标签现在允许我们用共价结合的脉冲标记蛋白质 并随着时间的推移追逐这些标记的蛋白质。这种活体脉搏追踪法已被用于 细胞培养中染色质重塑和小鼠肿瘤中蛋白质的稳定性。我们的初步结果表明 我们能够利用活体脉冲追踪法跟踪不同条件下虫种组织中组蛋白的稳定性。 营养条件。目前的目标是在体内使用线虫的脉冲追逐来可视化 母体生殖系RNA结合蛋白通过生殖细胞发育并跟踪这些蛋白的现状 从母亲传给后代的。首先，我们将脉冲标记一个P颗粒组装蛋白，并测试颗粒 形成和蛋白质含量影响其遗传。第二，我们将追踪母系Argavies的脉搏， 一个RNA调节酶家族，用来识别后代遗传了哪些宇航员，继承了什么组织 以及哪些蛋白质属性是组织特异性遗传所必需的。总的来说，这项工作将重新定义 我们的母系遗传概念，并阐明了特定RNA结合蛋白的遗传标准。在……里面 线虫体内的脉冲追逐将为发现新的母系遗传蛋白提供基础 与发育、免疫等方面的基因调控有关。 1