Germline Regulation by PUF/CPEB Protein Complexes

描述(申请人提供)：信使核糖核酸控制渗透生物学。许多转录本在其3‘非翻译区(3’UTRs)中含有顺式作用的调控特征1.这些元件招募能够调节运输、翻译和稳定性的调控因子2.多个RNA结合蛋白聚集在给定的3‘UTR上；通常彼此直接相互作用5.这种复合体是mRNA调控的主要主题，特别是在早期发育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.这项提议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体的关键方面，这些蛋白质涉及跨越早期发育、学习和记忆8-13的不同生物过程。我们选择研究CPEB(细胞质多聚腺苷化元件结合)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‘UTR14结合来调节mRNA的表达。大部分工作背后的假设是，PUF和CPEB蛋白之间的相互作用是由一个离散的分子界面介导的。我的初步数据支持这一观点：CPB-1中的一个40个氨基酸的肽和FBF-2中的一个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Wkins实验室35中开发的分析方法分离破坏或增强结合的突变。在第二个目的中，我们验证了CPB-1增强FBF-2与mRNA结合亲和力的假设。为此，我们使用了高通量测序策略，分析了有和没有CPB-1的FBF-2序列特异性。在第三个目标中，我们验证了CPB-1/FBF相互作用增强转录抑制的假设，这是体内精子发生所必需的。我们的初步数据表明，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试方法，在这种方法中，我们通过将短肽注入活的动物体内来破坏这种复合体。我的具体目标如下。目的1-确定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复合体的深度是创新的，我们使用的技术也是如此；包括用于评估RNA结合特异性的深度测序，以及用于评估体内功能的多肽注射策略。我们的研究虽然主要集中在PUF-CPEB的相互作用上，但也将广泛地涉及3‘UTR控制的机制。 公共卫生相关性：PUF蛋白与RNA结合，是干细胞更新、学习和记忆以及某些形式的再生所必需的20；CPEB还与RNA结合，并与癌症、学习和记忆有关8-13、15、37。这项建议的核心主题是阐明CPEB/PUF复合体的关键生化和生物学特征。在这样做的过程中，我们希望阐明调控蛋白复合体控制mRNA的广泛主题。

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. PUBLIC HEALTH RELEVANCE: PUF proteins bind RNA and are required for stem cell renewal, learning and memory, and certain forms of regeneration 20; CPEBs also bind RNA and have been implicated in cancer, learning, and memory 8-13,15,37. The core theme of this proposal is to elucidate key biochemical and biological features of CPEB/PUF complexes. In so doing, we hope to illuminate broad themes of mRNA control by regulatory protein complexes.