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 长期目标是阐明大分子RNA-蛋白质复合物 将信息传递给后代细胞和后代。我们通常认为DNA和DNA 修改作为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中具有功能， 免疫力我的实验室旨在确定哪些RNA结合蛋白是遗传的，确定大分子 组织遗传的RNA-蛋白质复合物，并发现遗传这些蛋白质的分子目的 配合物这种对遗传RNA结合蛋白的兴趣目前延伸到研究RNA-蛋白质 形成多组分RNA-蛋白质颗粒或生物分子缩合物的复合物。我以前的很多 研究工作集中在P颗粒的结构组织特征上，P颗粒是一种遗传的RNA-蛋白颗粒 必要的C。线虫生殖细胞发育。未来五年将重点关注理解 RNA-蛋白质复合物如何在细胞世代之间以及从父母到 后代哪些RNA结合蛋白是遗传的，哪些细胞会遗传这些蛋白？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现有条件的限制。 跟踪多细胞生物中蛋白质成分的方法。我的实验室试图标记并跟踪母亲 蛋白质在真实的C. elegans，一个被证明的模式生物，研究基本问题， 动物发展。已建立的单基因编辑方法，强大的成像能力， 世代时间使C.线虫是研究母体蛋白功能的理想多细胞动物 代代相传。经过修饰的酶标签现在允许我们脉冲标记蛋白质， 配体并随时间推移追踪这些标记的蛋白质。这种体内脉冲追踪方法已被用于跟踪 细胞培养中的染色质重塑和小鼠肿瘤中的蛋白质稳定性。我们的初步结果表明 我们能够使用体内脉冲追踪来追踪不同条件下蠕虫生殖系组织中组蛋白的稳定性， 营养条件。目前的目标是在C. elegans来可视化 母体生殖系RNA结合蛋白通过生殖细胞发育和跟踪这些蛋白质，因为它们是 从母亲遗传给后代。首先，我们将脉冲标记一个P颗粒组装蛋白，并测试颗粒 蛋白质含量影响其遗传。第二，我们将脉冲追踪母体生殖系Argonautes， RNA调节酶家族，以确定哪些Argonaute由后代遗传，哪些组织遗传 它们，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 我们的概念母系遗传和阐明标准的遗传特异性RNA结合蛋白。在 活体脉冲追踪法。线虫将为发现新的母系遗传蛋白质提供基础 与发育、免疫等基因调控有关。 1

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