Comprehensive identification of post transcriptional regulators of gld-1 mRNA

gld-1 mRNA转录后调节因子的全面鉴定

• 批准号: 8639363
• 负责人: John Brenner
• 金额: $ 5.33万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8639363
• 关键词: Accounting; Adopted; Affinity Chromatography; Bacteriophages; Binding; Binding Proteins; Biological Assay; Biotin; Caenorhabditis elegans; Cataloging; Catalogs; Cell Proliferation; Cells; Commit; Cytoplasmic Protein; Data; Distal; Ensure; Event; Failure; Gene Expression; Genes; Genetic; Genetic Translation; Goals; In Vitro; Maintenance; Mass Spectrum Analysis; Measures; Meiosis; Messenger RNA; MicroRNAs; Molecular; Molecular Models; Pathway interactions; Phenotype; Polynucleotide Adenylyltransferase; Post-Transcriptional Regulation; Post-Translational Protein Processing; Prophase; Protein Binding; Proteins; Publishing; RNA; RNA Binding; RNA Interference; RNA Sequences; RNA-Binding Proteins; Regulation; Regulator Genes; Reporting; Repression; Role; SWI1; Stem cells; Streptavidin; Testing; Translating; Translational Activation; Translational Regulation; Translational Repression; Translations; Validation; aptamer; base; crosslink; genetic analysis; in vivo; insight; molecular modeling; mutant; protein complex; public health relevance; self-renewal; stem cell differentiation; stem cell fate; translation factor; tumor

DESCRIPTION (provided by applicant): The central goal of this project is to comprehensively identify post-transcriptional regulators of gld-1, which encodes a conserved RNA-binding protein that acts to promote the decision for germline stem cells to differentiate and enter meiosis. Post-transcriptional regulation of gene expression contributes to the decision of a stem cell to either maintain the stem cell fate or to differentiate. In the germline stem cells of C. elegans, posttranscriptional repression of gld-1 contributes to maintenance of the stem cell fate whereas translational activation of gld-1 promotes meiotic entry. Multiple post-transcriptional regulators of gld-1 are known: FBF-1 and FBF-2 are RNA-binding proteins that directly repress gld-1, whereas the RNA binding protein GLD-3, the poly-A polymerases GLD-2 and GLD-4, and NOS-3 activate gld-1. However, genetic analysis of these genes indicates other regulators must also contribute to gld-1 repression and activation in the distal germline. We propose to use RNA-based affinity purification of proteins that bind to the gld-1 mRNA in vitro and in vivo, followed y mass spectrometry to identify these bound proteins. This unbiased approach will generate a comprehensive catalogue of proteins that bind to the gld-1 mRNA during translational repression and during translational activation. Identified proteins will be validated for a role in gld-1 repression or activation genetically. A genetics approach will also be used to determine if the microRNA pathway represses gld-1 in germline stem cells, which was hypothesized based on recent computational, genetic, and functional data. Identification and validation of new gld-1 regulators is necessary to generate a molecular model for how gld-1 switches from being repressed to being activated as part of the decision of germline stem cells to enter meiosis. Understanding this switch will provide important insight into how stem cells initiate intracellular events to execute the decision to differentiate.

描述（由申请人提供）：该项目的中心目标是全面鉴定gld-1的转录后调节因子，gld-1编码保守的RNA结合蛋白，其作用是促进生殖干细胞分化和进入减数分裂的决定。 基因表达的转录后调控有助于干细胞决定维持干细胞命运还是分化。在秀丽隐杆线虫的生殖干细胞中，gld-1 的转录后抑制有助于干细胞命运的维持，而 gld-1 的翻译激活则促进减数分裂进入。 多个转录后调节因子 gld-1的功能是已知的：FBF-1和FBF-2是直接抑制gld-1的RNA结合蛋白，而RNA结合蛋白GLD-3、多聚腺苷酸聚合酶GLD-2和GLD-4以及NOS-3激活gld-1。然而，对这些基因的遗传分析表明，其他调节因子也必定有助于远端种系中 gld-1 的抑制和激活。我们建议使用基于 RNA 的亲和纯化在体外和体内与 gld-1 mRNA 结合的蛋白质，然后进行质谱分析来鉴定这些结合的蛋白质。这种公正的方法将生成一个全面的蛋白质目录，这些蛋白质在翻译抑制和翻译激活期间与 gld-1 mRNA 结合。鉴定出的蛋白质将被验证其作用 gld-1 基因抑制或激活。遗传学方法还将用于确定 microRNA 通路是否抑制生殖干细胞中的 gld-1，这是根据最近的计算、遗传和功能数据假设的。新的gld-1调节剂的鉴定和验证对于生成一个分子模型是必要的，该模型可以解释gld-1如何从被抑制转变为被激活，作为生殖系干细胞进入减数分裂决定的一部分。了解这种开关将为了解干细胞如何启动细胞内 执行决策的事件要区分。