RNA recognition by maternal gene silencers in nematodes

线虫母体基因沉默子对 RNA 的识别

基本信息

批准号：
8010022
负责人：
Sean Patrick Ryder
金额：
$ 12.5万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-01-08 至 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8010022
关键词：
3&apos Untranslated Regions Adult Affinity Animals Anterior Arthritis Autoimmune Diseases Base Sequence Binding Binding Sites Biological Assay Biological Models Caenorhabditis elegans Cell Maintenance Cells Complex Consensus Consensus Sequence Contraceptive methods Defect Development Discrimination Distal Elements Embryo Embryonic Development Functional RNA Gametogenesis Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Genetic Translation Goals Homologous Gene Human Biology Human Development Immunity Immunoprecipitation In Vitro Indium Individual Infertility Inflammation Inflammatory KH Domain Knowledge Lead Learning Life Mammals Maternal Messenger RNA Measures Mental disorders Messenger RNA Methods Modeling Molecular Multiple Sclerosis Mutation Nematoda Neuraxis Nucleotides Oocytes Organism Pattern Phenotype Play Positioning Attribute Process Proteins RNA RNA Binding RNA Sequences RNA-Binding Proteins Regulation Reporter Rheumatoid Arthritis Role Site Specificity Staging Stem cells Stretching Testing Therapeutic Thermodynamics Transcript Transgenic Organisms Translations Uridine Vascularization Work Zinc Fingers base blastomere structure cell fate specification combat crosslink embryo stage 2 genetic regulatory protein glucagon-like peptide 1 in vivo mutant myelination nervous system disorder notch protein novel pleiotropism research study response stem zygote

项目摘要

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.

项目摘要：我实验室的主要目标是定义非编码元素的基础信使RNA序列定义了基因表达的差异调节。模型系统还早线虫秀丽隐杆线虫的胚胎发生。实验策略是确定参与识别非核苷酸的每个蛋白质的核苷酸结合特异性和组装机制使用定量体外方法编码元件。然后，与每个蛋白质相关的 mRNA 是使用交联免疫沉淀和/或 RNA 免疫沉淀和阵列独立鉴定。这使用转基因报告基因在活体动物中测试结合特异性的功能相关性规定。这种方法与标准正向遗传学逻辑相反，但它可以进行定量分析仅仅使用体内方法不可能理解 mRNA 的区分。长期目标是我的实验室的目标是描绘胚胎中 RNA 调节电路的完整接线图，并阐明控制母体 mRNA 翻译、定位和周转的调节机制。必要的第一实现这一目标的一步是确定每个调节蛋白的 RNA 靶标，并确定它们如何发挥作用一起选择特定的 mRNA 进行调节。在本提案中，我们重点关注在Notch/glp-1表达模式中的RNA结合蛋白胚胎（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工作与这些因素的 mRNA 识别相关的发现，这些因素主张合作和对抗相互作用驱动 glp-1 转录本的识别。这些结果引出了我们当前的假设：RNA 的占据 glp-1 3'-UTR 上的结合蛋白定义了其空间和时间表达模式。具体目标该提案中概述的将测试该模型，并确定每种蛋白质的新调控目标有助于这些蛋白质中每一种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。我们建议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着重要作用，包括胎盘分化、中枢神经系统、血管化和免疫。从该项目中汲取的经验教训可能会有所帮助了解导致炎症性疾病、神经和精神疾病的人类生物学，以及先天性发育异常。项目叙述：该提案描述了旨在理解一个过程的实验受精卵转变成多细胞动物。通过定义管理初始的监管流程的发展，有可能开发出新的策略来对抗不孕症和新型避孕药具方法。最后，胚胎发生过程中的 RNA 调节、炎症之间存在令人惊讶的相关性。反应和中枢神经系统的髓鞘形成。这项工作可能会带来新的突破多炎性关节炎，包括类风湿性关节炎和其他自身免疫性疾病，包括多发性关节炎硬化。