RNA recognition by maternal gene silencers in nematodes

线虫母体基因沉默子对 RNA 的识别

• 批准号: 8033737
• 负责人: Sean Patrick Ryder
• 金额: $ 30.26万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8033737
• 关键词: 3' Untranslated Regions; Adult; Affinity; Animals; Anterior; Base Sequence; Binding; Binding Sites; Biological Assay; Biological Models; Caenorhabditis elegans; Cell Maintenance; Cells; Complex; Consensus; Consensus Sequence; Defect; Development; Discrimination; Disease; 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; Inflammatory; KH Domain; Knowledge; Lead; Learning; Life; Mammals; Maternal Messenger RNA; Measures; Mental disorders; Messenger RNA; Methods; Modeling; Molecular; Mutation; Nematoda; Neuraxis; Nucleotides; Oocytes; Organism; Pattern; Phenotype; Play; Positioning Attribute; Process; Proteins; RNA; RNA Binding; RNA Sequences; RNA-Binding Proteins; Regulation; Reporter; 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; crosslink; embryo stage 2; genetic regulatory protein; glucagon-like peptide 1; in vivo; mutant; nervous system disorder; notch protein; novel; pleiotropism; research study; stem; zygote

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.

描述（由申请人提供）：我的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非编码元件识别的每种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鉴定与每种蛋白质相关的mRNA。使用分析调节的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。这种方法与标准正向遗传学的逻辑相反，但它能够定量理解 mRNA 区分，而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。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。在本提案中，我们重点关注胚胎中 Notch/glp-1 表达模式的 RNA 结合蛋白（MEX-3、MEX-5、POS-1、SPN-4 和 GLD-1）。在前期工作中，我们取得了一些与这些因素的 mRNA 识别相关的重要发现，这些因素认为合作和拮抗相互作用驱动 glp-1 转录本的识别。这些结果引出了我们当前的假设：glp-1 3'-UTR 上 RNA 结合蛋白的占据定义了其空间和时间表达模式。该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相关。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、血管化和免疫。从该项目中汲取的经验教训可能有助于了解导致炎症性疾病、神经和精神疾病以及先天性发育异常的人类生物学。