Comprehensive identification of post transcriptional regulators of gld-1 mRNA

描述（由申请人提供）：本项目的中心目标是全面鉴定gld-1的转录后调节因子，gld-1编码一种保守的RNA结合蛋白，其作用是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节有助于干细胞决定维持干细胞命运或分化。在C.在elegans中，GLD-1的转录后抑制有助于维持干细胞命运，而GLD-1的翻译激活促进减数分裂进入。 多种转录后调节因子 gld-1已知：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，其他调节因子也必须有助于gld-1在远端生殖系中的抑制和激活。我们建议使用基于RNA的亲和纯化的蛋白质，结合到gld-1 mRNA在体外和体内，然后y质谱鉴定这些结合的蛋白质。这种无偏见的方法将产生一个全面的目录的蛋白质结合的gld-1 mRNA在翻译抑制和翻译激活过程中。将验证鉴定的蛋白质在以下方面的作用： gld-1基因的抑制或激活。遗传学方法也将用于确定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，遗传和功能数据假设的。新的gld-1调节因子的鉴定和验证对于建立一个gld-1如何从被抑制转变为被激活的分子模型是必要的，这是生殖系干细胞决定进入减数分裂的一部分。了解这个开关将提供重要的洞察干细胞如何启动细胞内 事件来执行区分的决定。

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.