Germline Regulation by PUF/CPEB Protein Complexes

PUF/CPEB 蛋白复合物的种系调控

基本信息

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
项目类别：
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&apos 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

项目摘要

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 控制渗透到生物学中。许多转录本在其 3' 非翻译区 (3'UTR) 中含有顺式作用调节特征 1。这些元件招募能够调节运输、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。此类复合物是 mRNA 控制的主要主题，特别是在早期发育期间 6,7。我们的最终目标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关注涉及早期发育、学习和记忆等多种生物过程的一对相互作用的蛋白质来阐明这些复合物的关键方面8-13。我们选择研究 CPEB（细胞质多腺苷酸化元件结合）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。这项工作的大部分假设是 PUF 和 CPEB 蛋白之间的相互作用是由离散分子界面介导的。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，我们深入分析相互作用的分子基础，使用 Wickens 实验室 35 开发的测定法分离破坏或增强结合的突变。在第二个目标中，我们测试了以下假设：FBF-2 的结合亲和力CPB-1 可增强 mRNA。为此，我们使用高通量测序策略来分析有或没有 CPB-1 的 FBF-2 序列特异性。在第三个目标中，我们测试了这样的假设：CPB-1/FBF 相互作用增强了转换抑制，并且是体内精子发生所必需的。我们基于初步数据表明 CPB-1 在体外增强 FBF-2 的抑制。我们还描述了一种新的测定方法，通过将短肽注射到活体动物体内来破坏复合物。我的具体目标如下。目标 1 - 鉴定 FBF-2 和 CPB-1 中相互作用所需的残基。目标 2 - 确定 CPB-1 结合对 FBF-2 对 RNA 亲和力的影响。目标 3 - 阐明 CPB-1 与 FBF-2 结合的功能效应。我们研究 3'UTR 复合体的深度和我们使用的技术都是创新的；包括评估 RNA 结合特异性的深度测序，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，但将广泛涉及 3'UTR 控制机制。