RNA recognition by maternal gene silencers in nematodes

描述（由申请人提供）：我实验室的主要目标是确定信使RNA序列中的非编码元件定义基因表达差异调节的基础。该模型系统是线虫秀丽隐杆线虫的早期胚胎发生。实验策略是使用定量体外方法确定参与识别非编码元件的每个蛋白质的核苷酸结合特异性和组装机制。然后，使用交联免疫沉淀和/或RNA免疫沉淀和阵列独立地鉴定与每种蛋白质缔合的mRNA。使用测定调节的转基因报告基因在活动物中测试结合特异性的功能相关性。这种方法在逻辑上与标准的正向遗传学相反，但它能够定量地理解mRNA的区分，而这仅仅使用体内方法是不可能的。我实验室的长期目标是描绘胚胎中RNA调控回路的完整接线图，并阐明控制母体mRNA翻译，定位和周转的调控机制。实现这一目标的必要的第一步是确定每个调节蛋白的RNA靶点，并确定它们如何共同作用以选择特定的mRNA进行调节。在这项提案中，我们专注于在胚胎中模式Notch/glp-1表达的RNA结合蛋白（MEX-3，MEX-5，POS-1，SPN-4和GLD-1）。在初步工作中，我们已经取得了一些重要的发现，相关的mRNA识别的这些因素，认为合作和拮抗相互作用驱动识别的GLP-1转录本。这些结果导致了我们目前的假设：在glp-1 3 '-UTR上的RNA结合蛋白的占据决定了其时空表达模式。本提案中概述的具体目标将测试该模型，并确定每种蛋白质的新调控靶点，这些靶点可能有助于这些蛋白质中每种蛋白质的突变表型的多效性和差异。我们的工作将描述有助于胚胎细胞全能性的基本机制，这与几种现代治疗策略有关。我们打算研究的所有蛋白质在哺乳动物中都有同源物，其中许多蛋白质在人类发育中发挥作用，包括胎盘分化、中枢神经系统的形成、血管形成和免疫。从这个项目中吸取的经验教训可能有助于了解人类生物学，有助于炎症性疾病，神经和精神疾病，以及先天性发育异常。

DESCRIPTION (provided by applicant): The primary goal of my lab is to define the basis by which non-coding elements in messenger RNA sequences define differential regulation of gene expression. The model system is early embryogenesis of the nematode Caenorhabiditis elegans. The experimental strategy is to determine the nucleotide binding specificity and assembly mechanism of each protein involved in recognition of the noncoding elements using quantitative in vitro methods. Then, the mRNAs that associate with each protein are independently identified using crosslinked immunprecipitation and/or RNA-immunoprecipitation and array. The functional relevance of the binding specificity is tested in live animals using transgenic reporters that assay for regulation. This approach is the logical opposite of standard forward genetics, yet it enables a quantitative understanding of mRNA discrimination that is not possible using solely in vivo methods. The long term goal of my lab is to delineate the complete wiring diagram of RNA regulatory circuitry in the embryo, and elucidate the regulatory mechanisms that control maternal mRNA translation, localization, and turnover. A necessary first step toward this goal is to identify the RNA targets of each regulatory protein, and determine how they work together to select specific mRNAs for regulation. In this proposal, we focus on the RNA-binding proteins that pattern Notch/glp-1 expression in the embryo (MEX-3, MEX-5, POS-1, SPN-4, and GLD-1). In preliminary work, we have made a several important discoveries relevant to mRNA recognition by these factors that argue cooperative and antagonistic interactions drive recognition of glp-1 transcripts. These results lead to our current hypothesis: Occupancy of the RNA binding proteins on the glp-1 3'-UTR defines its spatial and temporal expression pattern. The specific aims outlined in this proposal will test this model, and identify novel regulatory targets of each protein that may contribute to the pleiotropy and disparity of the mutant phenotypes for each of these proteins. Our work will describe basic mechanisms that contribute to the totipotency of embryonic cells, which has relevance to several modern therapeutic strategies. All of the proteins that we propose to study have homologs in mammals, many of which play roles in human development, including placental differentiation, formation of the central nervous system, vascularization, and immunity. Lessons learned from this project may aid in understanding human biology that contributes to inflammatory disease, neurological and psychiatric disorders, and congenital developmental abnormalities.