RNA recognition by maternal gene silencers in nematodes

项目概述：我的实验室的主要目标是定义非编码元素在 信使RNA序列定义了基因表达的差异调节。模型系统早在 线虫的胚胎发生。实验策略是确定 核苷酸结合特异性和组装机制的每种蛋白参与识别的非- 使用体外定量方法测定编码元件。然后，与每种蛋白质相关的mRNA被 使用交联免疫沉淀和/或RNA免疫沉淀和阵列独立鉴定。的 在活动物中使用转基因报道分子测试结合特异性的功能相关性， 调控这种方法与标准的正向遗传学在逻辑上是相反的，但它能够定量地分析遗传学。 理解mRNA的区别，这是不可能的，只使用体内方法。的长期目标 我的实验室是描绘出胚胎中RNA调控回路的完整接线图，并阐明 控制母体mRNA翻译、定位和周转的调节机制。必要的第一 实现这一目标的第一步是确定每个调节蛋白的RNA靶点，并确定它们是如何工作的 共同选择特定的mRNA进行调控。 在这个建议中，我们专注于RNA结合蛋白的模式Notch/GLP-1的表达， 胚胎（MEX-3、MEX-5、POS-1、SPN-4和GLD-1）。在前期工作中，我们做了几个重要的 与这些因素识别mRNA相关的发现，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的占据- glp-13 ′-UTR上的结合蛋白决定了其空间和时间表达模式。具体目标 在这项提案中概述的将测试这个模型，并确定每种蛋白质的新的调控目标， 有助于这些蛋白质中的每一种的突变表型的多效性和差异性。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 几种现代治疗策略我们打算研究的所有蛋白质都有同源物， 哺乳动物，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、血管化和免疫。从该项目中吸取的经验教训可能有助于 了解导致炎症性疾病、神经和精神疾病的人类生物学， 和先天性发育异常项目叙述：该提案描述了旨在了解一个人的行为的过程的实验。 受精卵变成多细胞动物。通过定义管理初始 发展，它可能会开发新的战略，以打击不孕症和新的避孕 方法.最后，在胚胎发育过程中的RNA调节，炎症， 反应和中枢神经系统的髓鞘形成。这项工作可能会导致新的突破有关 包括类风湿性关节炎和其他自身免疫性疾病，包括多发性 硬化症

Project Summary: 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 non- coding 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. Project Narrative: This proposal describes experiments aimed at understanding the process by which a fertilized egg transforms into a multicellular animal. By defining the regulatory processes that govern initial development, it may be possible to develop new strategies to combat infertility and novel contraceptive methods. Lastly, there is a surprising correlation between RNA regulation during embryogenesis, inflammation response, and myelination in the central nervous system. This work may lead to new breakthroughs relevant to polyinflammatory arthritides including rheumatoid arthritis and other autoimmune disorders including multiple sclerosis.