Understanding how Vasa Proteins Promote Cellular Pluripotency in the C. elegans Germline (Childcare Costs Supplement)

项目概要/摘要 Vasa DEAD-box RNA 解旋酶可调节秀丽隐杆线虫等动物的种系多能性 人类。 Vasa 蛋白在发育过程中经常被重新用作瞬时多能因子， 再生和肿瘤发生。 Vasa 驱动多能性的分子机制尚不清楚。 果蝇的初步研究支持瓦萨在翻译中的作用，但最近的工作集中在它的 在小 RNA 生物合成中发挥作用并作为 RNA 溶剂。该项目的长期目标是确定 Vasa 蛋白如何促进细胞多能性以操纵其功能以增强再生或 缓慢的肿瘤发生。直接目标是检验线虫 Vasa 同源物的假设， GLH-1，调节内质网定向翻译。在秀丽隐杆线虫中，Vasa 同源物的 GLH 家族发挥作用 冗余，允许在不影响生育能力的情况下研究突变体。鉴定 GLH-1 结合 在体内合作伙伴中，我们使用定量质谱法从全线虫裂解液中分离出蛋白质 与 GLH-1 进行免疫共沉淀。结果强调了 GLH-1 和 tRNA 之间的关联 合成酶、翻译因子和 ER 易位子的多种成分。比较这些协会 对具有精确 GLH-1 突变的线虫的研究表明，易位子相互作用是由锌介导的 Vasa 蛋白特有的指结构域。在目标 1 中，我们将确定 GLH-1 与 使用体内分裂超级文件夹 GFP 策略进行易位。在目标 2 中，我们将通过以下方式测试功能交互： 将精确的突变引入易位子并测量 GLH-1 对所得 ER 的调节 功能障碍。在目标 3 中，我们将检验 GLH-1 调节 ER 定向转录本翻译的假设 通过对野生型和 GLH-1 缺失蠕虫进行多核糖体分析。拟议的实验将 揭示生殖细胞生物学的新方面以及 Vasa 发挥功能的新转录后机制 在种系中。他们的完成将导致在 Vasa 转录后鉴定下的转录本 控制，这将代表一组新的治疗靶点，以增强再生并减缓肿瘤发生。 申请人 Emily Spaulding 博士将在 Mount Desert Island Biological 进行该研究项目 位于缅因州巴港的实验室 (MDIBL)。 MDIBL拥有122年的生物医学研究历史，是NIH 再生生物学和医学卓越生物医学研究中心。申请人的担保人， 达斯汀·厄普代克 (Dustin Updike) 博士是美国国立卫生研究院 (NIH) 资助的副教授，拥有良好的成功记录 线虫生殖细胞生物学方面的指导和卓越表现。执行斯波尔丁博士的培训计划将增加 线虫研究和生殖细胞生物学方面的专业知识与她在小鼠翻译调控方面的背景相结合。 拟议的培训支持 Spaulding 博士的目标，即运营一个利用以下优势的实验室： 线虫和小鼠研究不同细胞类型如何以独特的方式代谢 RNA 以及如何 这些机制的扰动可能导致人类疾病。

PROJECT SUMMARY/ABSTRACT Vasa DEAD-box RNA helicases regulate germline pluripotency in animals ranging from C. elegans to humans. Vasa proteins are frequently repurposed as transient pluripotency factors during development, regeneration, and tumorigenesis. The molecular mechanisms by which Vasa drives pluripotency are unclear. Initial studies in Drosophila supported a role for Vasa in translation, but more recent work has focused on its functions in small RNA biogenesis and as an RNA solvent. The long-term goal of this project is to determine how Vasa proteins promote cellular pluripotency in order to manipulate its function to enhance regeneration or slow tumorigenesis. The immediate objective is to test the hypothesis that the C. elegans Vasa homolog, GLH-1, regulates ER-directed translation. In C. elegans, the GLH family of Vasa homologs function redundantly, allowing for the study of mutants without compromised fertility. To identify GLH-1 binding partners in vivo we have used quantitative mass spectrometry to isolate proteins from whole worm lysate that co-immunoprecipitate with GLH-1. The results emphasize associations between GLH-1 and tRNA synthetases, translation factors, and multiple components of the ER translocon. Comparing these associations to those in worms with precise GLH-1 mutations reveals that translocon interactions are mediated by a zinc finger domain unique to Vasa proteins. In Aim 1 we will determine GLH-1’s spatiotemporal association with the translocon using an in vivo split-superfolder GFP strategy. In Aim 2 we will test for a functional interaction by introducing precise mutations into the translocon and measuring GLH-1’s modulation of resulting ER dysfunction. In Aim 3 we will test the hypothesis that GLH-1 regulates the translation of ER-directed transcripts by performing polysome profiling of wild-type and GLH-1 deletion worms. The proposed experiments will reveal a new aspect of germ cell biology and a novel post-transcriptional mechanism by which Vasa functions in the germline. Their completion will result in identification of transcripts under Vasa post-transcriptional control, which will represent a new set of therapeutic targets to enhance regeneration and slow tumorigenesis. The applicant, Dr. Emily Spaulding, will perform the research project at Mount Desert Island Biological Laboratory (MDIBL) in Bar Harbor, Maine. MDIBL has a 122-year history of biomedical research and is an NIH Center for Biomedical Research Excellence in regenerative biology and medicine. The applicant’s sponsor, Dr. Dustin Updike, is an NIH-funded associate professor and has a well-documented record of successful mentorship and excellence in C. elegans germ cell biology. Execution of Dr. Spaulding’s training plan will add expertise in C. elegans research and germ cell biology to her background in translational regulation in mice. The proposed training supports Dr. Spaulding’s goal of running a laboratory that leverages the advantages of both C. elegans and the mouse to investigate how distinct cell types metabolize RNA in unique ways and how perturbation of these mechanisms can lead to human disease.