Germline Regulation by PUF/CPEB Protein Complexes

PUF/CPEB 蛋白复合物的种系调控

• 批准号: 8370566
• 负责人: Zachary Campbell
• 金额: $ 5.39万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370566
• 关键词: 3' Untranslated Regions; Affinity; Amino Acids; Animal Model; Animals; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Process; Biology; CPE-binding protein; Caenorhabditis elegans; Cells; Complex; Data; Development; Elements; Family; Genetic Translation; In Vitro; Injection of therapeutic agent; Learning; Life; Malignant Neoplasms; Mediating; Memory; Messenger RNA; Molecular; Mutation; N-terminal; Natural regeneration; Oocytes; Oogenesis; Pattern Formation; Peptides; Polyadenylation; Polynucleotide Adenylyltransferase; Proliferating; Proteins; RNA; RNA Binding; RNA Interference; RNA Recognition Motif; RNA Sequences; RNA-Binding Proteins; Recruitment Activity; Regulation; Repression; Research; Role; Specificity; Spermatocytes; Spermatogenesis; Stem cells; Techniques; Testing; Transcript; Translating; Translations; Ursidae Family; Work; Zinc; base; combinatorial; genetic regulatory protein; in vivo; innovation; mRNA Decay; mRNA Expression; member; protein complex; sperm cell; stem cell division; tumor

DESCRIPTION (provided by applicant): mRNA control permeates biology. Many transcripts contain cis-acting regulatory features in their 3' untranslated regions (3'UTRs) 1. These elements recruit regulatory factors capable of modulating transport, translation, and stability 2,3. Multiple RNA-binding proteins congregate onto a given 3'UTR; often interacting directly with one another 5. Such complexes are a dominant theme in mRNA control particularly during early development 6,7. Our ultimate aim is to characterize the molecular and structural mechanisms promoting assembly of regulatory proteins onto the 3'UTR. The core theme of this proposal is to elucidate key aspects of these complexes by focusing on a pair of interacting proteins implicated in diverse biological processes spanning early development, learning, and memory 8-13. We have chosen to study the interaction between a CPEB (Cytoplasmic Polyadenylation Element Binding) and PUF (Pumilio and FBF) protein. Members of these two families collaborate to regulate mRNA expression via binding to the 3' UTR 14. The hypothesis underlying much of this work is that the interaction between PUF and CPEB proteins is mediated by a discrete molecular interface. My preliminary data support this idea: a 40 amino acid peptide in CPB-1 and a short loop in FBF-2 are required for their interaction. In the first aim, we analyze the molecular basis of the interaction in depth, isolating mutations that disrupt or enhance binding using assays developed in the Wickens lab 35. In the second aim, we test the hypothesis that the binding affinity of FBF-2 for mRNA is enhanced by CPB-1. To do so, we use a high-throughput sequencing strategy analyzing FBF-2 sequence specificity with and without CPB-1. In the third aim, we test the hypothesis that the CPB-1/FBF interaction enhances transational repression, and is required for spermatogenesis in vivo. We build on preliminary data that suggest CPB-1 enhances repression by FBF-2 in vitro. We also describe a new assay in which we disrupt the complex by injection of short peptides into living animals. My specific aims are as follows. Aim 1 - To identify residues in FBF-2 and CPB-1 required for their interaction. Aim 2 - To determine the effects of CPB-1 binding on the affinity of FBF-2 for RNA. Aim 3 - To elucidate the functional effects of CPB-1 binding to FBF-2. The depth in which we will study a 3'UTR complex is innovative as are the techniques we use; including deep sequencing to assess RNA binding specificity, and a peptide injection strategy to assess function in vivo. Our research, while focused tightly on the PUF-CPEB interaction, will bear broadly on mechanisms of 3'UTR control.

描述（由申请人提供）：mRNA对照渗透生物学。许多转录物在其3'非翻译区（3' UTR）中含有顺式作用调控特征1。这些元素招募能够调节运输，翻译和稳定性的调节因子2，3。多个RNA结合蛋白聚集到给定的3 'UTR上;通常彼此直接相互作用5。这种复合物是mRNA控制的主导主题，特别是在早期发育期间6，7。我们的最终目的是表征促进调控蛋白组装到3 'UTR上的分子和结构机制。该提案的核心主题是通过关注一对相互作用的蛋白质来阐明这些复合物的关键方面，这些蛋白质涉及跨越早期发育，学习和记忆的各种生物过程8-13。我们选择研究CPEB（细胞质多聚腺苷酸化元件结合）和PUF（Pumilio和FBF）蛋白之间的相互作用。这两个家族的成员通过与3' UTR 14结合来协作调节mRNA表达。这项工作的基础假设是PUF和CPEB蛋白之间的相互作用是由离散的分子界面介导的。我的初步数据支持这个想法：CPB-1中的40个氨基酸的肽和FBF-2中的短环是它们相互作用所必需的。在第一个目标中，我们深入分析了相互作用的分子基础，使用Wickens实验室开发的检测方法分离破坏或增强结合的突变35。在第二个目标中，我们测试了这样的假设，即CPB-1增强了FBF-2对mRNA的结合亲和力。为此，我们使用高通量测序策略分析有和没有CPB-1的FBF-2序列特异性。在第三个目标中，我们测试的假设，CPB-1/FBF相互作用增强transational抑制，并需要在体内精子发生。我们建立在初步的数据表明，CPB-1增强抑制FBF-2在体外。我们还描述了一种新的检测方法，在该方法中，我们通过将短肽注射到活体动物中来破坏复合物。我的具体目标如下。 目的1 -鉴定FBF-2和CPB-1相互作用所需的残基。 目的2 -确定CPB-1结合对FBF-2与RNA亲和力的影响。 目的3 -阐明CPB-1与FBF-2结合的功能效应。我们将研究3 'UTR复合物的深度是创新的，我们使用的技术也是创新的;包括深度测序以评估RNA结合特异性，以及肽注射策略以评估体内功能。我们的研究，同时密切关注PUF-CPEB相互作用，将广泛承担3 'UTR控制的机制。