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

项目摘要/摘要 VASA死盒RNA解旋酶调节从线虫到线虫等动物的生殖系多能性 人类。VASA蛋白在发育过程中经常被改变用途，作为瞬时多能性因子， 再生和肿瘤形成。VASA驱动多能性的分子机制尚不清楚。 最初对果蝇的研究支持Vasa在翻译中的作用，但最近的工作集中在它的 在小RNA生物合成中的作用以及作为RNA溶剂。这个项目的长期目标是确定 VASA蛋白如何促进细胞的多能性以操纵其功能以促进再生或 缓慢的肿瘤形成。直接的目标是测试线虫瓦萨同源线虫的假设， GLH-1，调节内质网导向的翻译。在线虫中，Vasa同源基因的GLH家族的功能 多余的，允许在不影响生育能力的情况下研究突变。识别GLH-1结合 在活体中，我们使用定量质谱学从整个虫子裂解液中分离出蛋白质 与GLH-1免疫共沉淀。结果强调了GLH-1和tRNA之间的联系 合成酶、翻译因子和内质网转位蛋白的多种成分。比较这些关联 对那些带有精确GLH-1突变的蠕虫的研究表明，转位基因的相互作用是由锌介导的 Finger结构域是Vasa蛋白所特有的。在目标1中，我们将确定GLH1‘S与 使用体内分裂超文件夹GFP策略的易位子。在目标2中，我们将通过以下方式测试功能交互 在易位基因中引入精确突变并检测GLH-1‘S对产生的内质网的调节 功能障碍。在目标3中，我们将检验GLH-1调节ER导向转录本的翻译的假设 通过对野生型和GLH-1缺失型蠕虫进行多聚体分析。拟议中的实验将 揭示生殖细胞生物学的新方面和VASA发挥作用的新的转录后机制 在生殖系中。它们的完成将导致在VASA转录后识别抄本 对照，这将代表一套新的治疗目标，以促进再生和减缓肿瘤形成。 申请者艾米丽·斯波尔丁博士将在荒山岛生物研究所进行这项研究项目 缅因州巴尔港的实验室(MDIBL)。MDIBL拥有122年的生物医学研究历史，是美国国立卫生研究院的 再生生物学和医学方面的卓越生物医学研究中心。申请人的保证人， 达斯汀·厄普代克博士是美国国立卫生研究院资助的副教授，有很好的成功记录 在线虫生殖细胞生物学方面的指导和卓越表现。执行Spaulding博士的培训计划将增加 她在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吻合。 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，研究不同类型的细胞如何以独特的方式代谢RNA以及如何 这些机制的扰动可能会导致人类疾病。

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.