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控制渗透到生物学。许多转录本在其3'未翻译区域（3'UTRS）1中包含顺式作用调节特征1。这些元素募集了能够调节运输，翻译和稳定性的调节因素2,3。多种RNA结合蛋白聚集在给定的3'UTR上；经常直接与一个人相互作用5。这种复合物是mRNA控制中的主要主题，尤其是在早期开发中6,7。我们的最终目的是表征促进调节蛋白组装到3'UTR上的分子和结构机制。该提案的核心主题是通过专注于涉及早期发展，学习和记忆的多种生物学过程的一对相互作用蛋白质来阐明这些复合物的关键方面。我们选择研究CPEB（细胞质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结合来调节mRNA表达。这项工作的大部分假设是PUF和CPEB蛋白之间的相互作用是由离散的分子界面介导的。我的初步数据支持了这一想法：CPB-1中的40个氨基酸肽和FBF-2中的短环是其相互作用所必需的。在第一个目的中，我们分析了深度相互作用的分子基础，分离出使用Wickens Lab 35中开发的测定方法破坏或增强结合的突变。在第二个目标中，我们检验了FBF-2对mRNA的结合亲和力通过CPB-1增强的假设。为此，我们使用有或没有CPB-1的FBF-2序列特异性分析FBF-2序列特异性的高通量测序策略。在第三个目的中，我们检验了CPB-1/FBF相互作用增强了横向抑制的假设，并且是体内精子发生所必需的。我们建立在初步数据的基础上，这表明CPB-1在体外增强了FBF-2的抑制作用。我们还描述了一种新的测定法，其中我们通过将短肽注射到活动物中来破坏复合物。我的具体目标如下。 目标1-确定其相互作用所需的FBF-2和CPB-1中的残基。 AIM 2-确定CPB-1结合对FBF-2对RNA的亲和力的影响。 AIM 3-阐明CPB-1与FBF-2结合的功能效应。我们研究3'UTR综合体的深度与我们使用的技术一样具有创新性。包括深层测序以评估RNA结合特异性，以及评估体内功能的肽注射策略。我们的研究虽然紧密关注PUF-CPEB相互作用，但将广泛地支持3'UTR控制的机制。