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

了解 Vasa 蛋白如何促进线虫种系的细胞多能性

基本信息

批准号：
10312175
负责人：
Emily Louisa Spaulding
金额：
$ 6.6万
依托单位：
MOUNT DESERT ISLAND BIOLOGICAL LAB
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-23 至 2023-08-22
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10312175
关键词：
Amino Acyl-tRNA Synthetases Animals Binding Biogenesis Biological Biomedical Research Caenorhabditis elegans Cellular biology Centers of Research Excellence Charge Cleaved cell Cytoplasm Cytoplasmic Granules Data Defect Development Drosophila genus Family Fertility Functional disorder Funding Genetic Transcription Germ Germ Cells Glycine Goals Homologous Gene Human Impairment Island Laboratories Lead Length Maine Mass Spectrum Analysis Measures Mediating Membrane Proteins Mentorship Modeling Molecular Mus Mutation Natural regeneration Nuclear Envelope Pathway interactions Peptide Initiation Factors Peptides Phenotype Phenylalanine Polyribosomes Post-Transcriptional Regulation Protein Family Proteins RNA RNA Helicase Recording of previous events Regenerative Medicine Regulation Research Research Project Grants Resolution Ribosomes Role Running Site Small RNA Solvents Testing Training Training Support Transcript Translational Regulation Translations Travel Tryptophan United States National Institutes of Health Work Zinc Fingers cell type experimental study falls helicase human disease in vivo insight mid-career faculty mutant novel pluripotency pluripotency factor polysome profiling protein folding regenerative biology response secretory protein spatiotemporal therapeutic target translation factor tumorigenesis

项目摘要

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.

项目摘要/摘要 VASA死盒RNA解旋酶调节从秀丽隐杆线虫到秀丽隐杆线到的动物种系多能人类。在开发过程中，经常将VASA蛋白重新用于瞬态多能因素，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了VASA在翻译中的作用，但最近的工作重点是在小的RNA生物发生和RNA溶液中起作用。该项目的长期目标是确定 VASA蛋白如何促进细胞多能力以操纵其功能以增强再生或缓慢的肿瘤发生。直接目的是检验以下假设：秀丽隐杆线虫VASA同源物， GLH-1，调节ER指导的翻译。在秀丽隐杆线虫中，vasa同源物功能的GLH家族冗余，允许研究突变体而不会受到损害的生育能力。识别GLH-1结合在体内的伴侣我们已经使用定量质谱法将蛋白质与整个蠕虫裂解物分离出来与GLH-1共免疫沉淀。结果强调了GLH-1与tRNA之间的关联 ER转运的合成酶，翻译因子和多个成分。比较这些关联对于具有精度GLH-1突变的蠕虫的人来说，易位相互作用是由锌介导的 VASA蛋白独有的手指域。在AIM 1中，我们将确定GLH-1与使用体内拆分 - 苏福利特GFP策略的转运。在AIM 2中，我们将通过将精确的突变引入转运，并测量GLH-1对产生的ER的调制功能障碍。在AIM 3中，我们将测试GLH-1调节ER指导转录本的翻译的假设通过对野生型和GLH-1删除蠕虫进行多元化分析。提出的实验将揭示生物细胞生物学的新方面和一种新型的转录后机制在种系中。他们的完成将导致在转录后的VASA下识别成绩单对照，这将代表一组新的治疗靶标，以增强再生和缓慢的肿瘤发生。申请人Emily Spaulding博士将在Mount Desert Island生物学进行研究项目缅因州巴港的实验室（MDIBL）。 MDIBL拥有122年的生物医学研究史，是NIH 再生生物学与医学卓越生物医学研究中心。申请人的赞助商，达斯汀·厄普克（Dustin Updike）博士是一位由NIH资助的副教授，并有据可查的成功记录 C.秀丽隐杆线虫生物细胞生物学的能态和卓越。执行Spaulding博士的培训计划将增加秀丽隐杆线虫研究和生殖细胞生物学的专业知识，在小鼠翻译调节方面的背景。拟议的培训支持Spaulding博士运营实验室的目标，该实验室利用了秀丽隐杆线虫和小鼠都研究了不同的细胞类型如何以独特的方式代谢RNA，以及这些机制的扰动会导致人类疾病。