Understanding how Vasa Proteins Promote Cellular Pluripotency in the C. elegans Germline

项目总结/摘要 Vasa DEAD-box RNA解旋酶调节动物的生殖系多能性，从C。优雅地 人类Vasa蛋白在发育过程中经常被重新利用为瞬时多能性因子， 再生和肿瘤发生。Vasa驱动多能性的分子机制尚不清楚。 对果蝇的初步研究支持Vasa在翻译中的作用，但最近的工作集中在它的 在小RNA生物合成中起作用并作为RNA溶剂。本项目的长期目标是确定 Vasa蛋白如何促进细胞多能性，以操纵其功能来增强再生， 肿瘤形成缓慢。当前的目标是检验C.线虫Vasa同源物， GLH-1，调节ER定向翻译。In C.线虫，GLH家族的Vasa同源物功能 冗余，允许研究突变体而不损害生育能力。鉴定GLH-1结合 体内合作伙伴我们使用定量质谱法从整个蠕虫裂解物中分离蛋白质， 与GLH-1共免疫沉淀。结果强调了GLH-1和tRNA之间的关联 合成酶、翻译因子和ER易位子的多种组分。比较这些关联 与GLH-1精确突变的蠕虫中的那些相比，揭示了易位相互作用是由锌介导的， Vasa蛋白质特有的指状结构域。在目标1中，我们将确定GLH-1的时空关联与 使用体内分裂-超级折叠GFP策略，使用一个易位子。在目标2中，我们将通过以下方式测试功能交互： 将精确的突变引入转座子并测量GLH-1对所产生的ER的调节 功能障碍在目标3中，我们将检验GLH-1调节ER定向转录物翻译的假设 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 揭示了生殖细胞生物学的一个新方面和Vasa功能的一种新的转录后机制 在生殖细胞中。它们的完成将导致在Vasa转录后水平下鉴定转录本。 控制，这将代表一组新的治疗靶点，以增强再生和减缓肿瘤发生。 申请人，艾米丽斯波尔丁博士，将在山荒岛生物执行研究项目 位于缅因州巴尔港的MDIBL实验室。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， 博士达斯汀·厄普代克是美国国立卫生研究院资助的副教授， 在C.生殖细胞生物学执行斯波尔丁博士的训练计划 专业知识C。elegans研究和生殖细胞生物学，她的背景是小鼠的翻译调控。 Spaulding博士的目标是运行一个利用以下优势的实验室， 两种C. elegans和小鼠研究不同的细胞类型如何以独特的方式代谢RNA， 这些机制的紊乱可导致人类疾病。

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.