Comprehensive identification of post transcriptional regulators of gld-1 mRNA

描述(申请人提供)：这个项目的中心目标是全面识别GLD-1的转录后调控因子，它编码一种保守的RNA结合蛋白，促进生殖系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是维持干细胞的命运还是分化。在线虫生殖系干细胞中，转录后抑制GLD-1有助于维持干细胞命运，而翻译激活GLD-1促进减数分裂进入。多个转录后调控因子 已知的GLD-1蛋白有：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、聚A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，在远端生殖系中，其他调控因子也必须对GLD-1的抑制和激活起作用。我们建议采用基于RNA的亲和纯化方法在体外和体内对与GLD-1mRNA结合的蛋白质进行亲和纯化，然后用质谱仪对这些结合蛋白进行鉴定。这种不偏不倚的方法将产生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-1mRNA结合。已识别的蛋白质将被验证在 GLD-1基因的抑制或激活。遗传学方法也将被用来确定microRNA途径是否抑制生殖系干细胞中的GLD-1，这是基于最近的计算、遗传和功能数据而假设的。鉴定和验证新的GLD-1调节子是必要的，以产生一个分子模型，作为生殖系干细胞进入减数分裂的决定的一部分，GLD-1如何从抑制转换到激活。理解这一转变将为干细胞如何启动细胞内提供重要的见解 执行差异化决策的事件。

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.