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-蛋白质复合物的分子机制 将信息传递给后代细胞和后代。我们传统上认为 DNA 和 DNA 修改是细胞之间继承的唯一信息。最近的工作表明 RNA 和 RNA- 结合蛋白也是遗传的，这些蛋白在生物体发育和 免疫力。我的实验室旨在鉴定哪些 RNA 结合蛋白是遗传性的，确定大分子 遗传性RNA-蛋白质复合物的组织，并发现遗传这些蛋白质的分子目的 复合物。目前对遗传性 RNA 结合蛋白的兴趣已扩展到研究 RNA 蛋白 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 工作重点是表征 P 颗粒（一种遗传性 RNA 蛋白质颗粒）的结构组织 线虫生殖细胞发育所必需的。未来五年将重点关注了解 分子机制 RNA-蛋白质复合物如何在细胞世代之间以及从亲代遗传到 后代。哪些 RNA 结合蛋白是遗传的，哪些细胞继承了这些蛋白？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可用的限制 追踪多细胞生物中蛋白质成分的方法。我的实验室致力于标记并跟踪母亲 秀丽隐杆线虫真实种系组织中的蛋白质，这是一种经过验证的模式生物，可用于研究基本问题 动物发育。已建立的单基因编辑方法、强大的成像能力和短时间 世代时间使线虫成为鉴定母体蛋白质功能的理想多细胞动物 代代相传。修饰的酶标签现在允许我们用共价结合脉冲标记蛋白质 配体并随着时间的推移追逐这些标记的蛋白质。这种体内脉冲追踪方法已用于跟踪 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种系组织中组蛋白的稳定性 营养条件。目前的目标是使用线虫体内脉冲追踪来可视化稳定性 母体种系 RNA 结合蛋白通过生殖细胞发育并追踪这些蛋白质的原样 从母亲遗传给后代。首先，我们将对 P 颗粒组装蛋白进行脉冲标记并测试颗粒如何 形成和蛋白质数量影响其遗传。其次，我们将追踪母系阿尔戈英雄， RNA 调节酶家族，用于识别哪些 Argonautes 是由后代遗传的，哪些组织继承了哪些 它们，以及组织特异性遗传所必需的蛋白质属性。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 线虫体内脉冲追踪将为发现新型母系遗传蛋白奠定基础 与发育、免疫等基因调控相关。 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