RNA recognition by maternal gene silencers in nematodes

项目摘要：我的实验室的主要目标是定义 信使RNA序列定义了基因表达的差异调节。模式体系还很早 线虫线虫的胚胎发生。实验策略是确定 参与识别非核糖核酸的每种蛋白质的核苷酸结合特异性和组装机制 使用体外定量方法对元件进行编码。然后，与每种蛋白质相关的mRNAs是 使用交联式免疫沉淀和/或RNA免疫沉淀和阵列独立鉴定。这个 在活体动物中使用转基因报告对结合特异性的功能相关性进行测试 监管。这种方法与标准正向遗传学在逻辑上是相反的，但它使得 仅用活体方法是不可能理解信使核糖核酸识别的。的长期目标是 我的实验室是描绘胚胎中RNA调节电路的完整接线图，并阐明 控制母体mRNA翻译、本地化和周转的调控机制。必要的第一步 朝着这个目标迈进的一步是确定每个调节蛋白的RNA靶标，并确定它们是如何工作的 共同选择特定的mRNAs进行调控。 在这个建议中，我们集中在RNA结合蛋白上，它使Notch/GLP-1在 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 与这些认为合作和拮抗相互作用的因素识别信使核糖核酸相关的发现 驱动对GLP-1转录本的识别。这些结果导致了我们目前的假设：RNA- GLP-1 3‘-UTR上的结合蛋白决定了其空间和时间表达模式。具体目标 这项提案中概述的将测试这一模型，并确定每种蛋白质的新调控靶点，可能 导致每种蛋白质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。我们计划研究的所有蛋白质在 哺乳动物，其中许多在人类发育中发挥作用，包括胎盘分化，形成 中枢神经系统、血管形成和免疫。从这个项目中学到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， 以及先天发育异常。项目说明：本提案描述了旨在理解以下过程的实验 受精卵转化为多细胞动物。通过定义管理初始阶段的监管流程 发展，就有可能开发抗击不孕症的新策略和新的避孕药 方法：研究方法。最后，在胚胎发生、炎症过程中的rna调控之间存在着令人惊讶的相关性。 反应和中枢神经系统的髓鞘形成。这项工作可能会带来相关的新突破 包括类风湿性关节炎在内的多炎性关节炎和其他自身免疫性疾病 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.