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控的基础。模型系统为秀丽隐杆线虫的早期胚胎发育。实验策略是利用体外定量方法确定参与非编码元件识别的每个蛋白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鉴定与每个蛋白质相关的mrna。结合特异性的功能相关性在活体动物中使用转基因报告基因检测调节。这种方法在逻辑上与标准正向遗传学相反，但它能够定量理解mRNA的区别，这是单独使用体内方法无法做到的。我的实验室的长期目标是描绘胚胎中RNA调控电路的完整接线图，并阐明控制母体mRNA翻译，定位和周转的调控机制。实现这一目标的第一步是确定每个调节蛋白的RNA靶标，并确定它们如何协同工作以选择特定的mrna进行调节。在本研究中，我们重点研究了胚胎中Notch/glp-1表达模式的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，这些因素与mRNA识别有关，这些因素认为合作和拮抗相互作用驱动glp-1转录物的识别。这些结果导致了我们目前的假设：RNA结合蛋白在glp-1 3'-UTR上的占用决定了其空间和时间表达模式。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，这些靶点可能导致每种蛋白质的多效性和突变表型的差异。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相关。我们拟研究的所有蛋白质在哺乳动物中都有同源物，其中许多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理解导致炎症性疾病、神经和精神疾病以及先天性发育异常的人类生物学。

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.