Structural Diversity of RNA-Binding Proteins

描述：（由申请人提供）RNA-蛋白质相互作用是核心 一个重要的目标是了解 决定其特异性的分子细节。以前的许多工作都集中在 在小模型系统中，来自病毒的富含精氨酸的肽 调节蛋白（包括HIV- 1达特和Rev以及BIV达特）识别它们的 RNA发夹靶向在周围蛋白质框架的情况下。的 结果表明，富含精氨酸的肽可以采用非常不同的二级结构， 结构，并且通常需要RNA的框架进行折叠。更好地 了解氨基酸-RNA相互作用如何促进RNA结合 不同背景下的特异性，从小的肽-RNA复合物到大的 多蛋白复合物，我们现在建议：1）使用组合策略， 更精确地定义氨基酸-RNA相互作用的分子细节， 模型系统和识别新的相互作用，以及2）定义RNA结合如何 特异性是在多蛋白的背景下建立和调节的， 复合物在体内，使用3'剪接处的分支点序列的识别 网站作为一个模型系统。具体目标1 wifi利用丰富的甜菜碱和锌 手指基序来产生组合文库，并将使用细菌 抗终止系统，以鉴定与RRE RNA和突变体结合的肽 该目的还通过实验测试氨基酸-碱基对相互作用 通过计算机预测，并且特别是涉及以下的烟碱-GU相互作用： 三个氢键和Rev-RRE的特异性变体改变 互动具体目标2将检查如何确定特异性， 哺乳动物分支点序列在3'剪接位点，在一个 涉及SF 1/mBBP的多蛋白复合物。在分支点和U2 AF处绑定 结合在邻近的多聚嘧啶段和AG二核苷酸上。氨基酸 对于分支点识别重要SF 1/mBBP的KB结构域将是 使用Tat-杂交系统鉴定，其中蛋白质融合到激活的 HIV-1达特的结构域被递送到HIV报告质粒中的RNA位点，并且 将尝试产生特异性改变突变体。Tat Hybrid 系统也将用于从cDNA文库中鉴定其他蛋白质， 可以改变分支点序列识别，或者可以与 SF 1/mBBP的选择性剪接形式。求婚的人。预计目标将增加 对氨基酸如何与RNA特异性相互作用的基本理解 以及不同的蛋白质环境如何改变这些识别特性， 在SF 1/mBBP模型系统中，3'剪接位点的选择受到一定影响。

DESCRIPTION: (Provided by Applicant) RNA-protein interactions are at the heart of many cellular processes, and an important goal is to understand the molecular details that govern their specificity. Much previous work has focused on small model systems in which arginine-rich peptides derived from viral regulatory proteins (including HIV- 1 Tat and Rev and BIV Tat) recognize their RNA hairpin targets in the absence of a surrounding protein framework. The results show that arginine-rich peptides can adopt very different secondary structures and often require the framework of the RNA for folding. To better understand how amino acid-RNA interactions contribute to RNA-binding specificity in different contexts, from small peptide-RNA complexes to large multiprotein complexes, we now propose to: 1) use combinatorial strategies to more precisely define molecular details of amino acid-RNA interactions in small model systems and identify novel interactions, and 2) define how RNA-binding specificity is established and regulated in the context of multiprotein complexes in vivo, using recognition of branchpoint sequences at 3' splice sites as a modeI system. Specific Aim 1 wifi utilize the arginine-rich and zinc fmger motifs to generate combinatorial libraries and will use a bacterial antitermination system to identify peptides that bind to the RRE RNA and mutant sites; This Aim also wifi experimentally test amino acid-base pair interactions predicted by computer, and in particular an arginine-GU interaction involving three hydrogen bonds and an altered specificity variant of the Rev-RRE interaction. Specific Aim 2 will examine how specificity is determined for mamnialian branchpoint sequences at 3' splice sites, in the context of a multiprotein complex involving SF1/mBBP. bound at the branchpoint and U2AF bound at an adjacent polypyrimidine tract and AG dincucleotide. Amino acids in the KB domain of SF1/mBBP important for branchpoint recognition will be identified using a Tat-hybrid system in which proteins fused to the activation domain of HIV-1 Tat are delivered to RNA sites in HIV reporter plasmids, and attempts will be made to generate change-of-specificity mutants. The Tat-hybrid system also will be used to identify other proteins from cDNA libraries that may alter branchpoint sequence recognition or that may interact with alternatively spliced forms of SF1/mBBP. The proposed. Aims are expected to add to the basic understanding of how amino acids interact specificially with RNAs and how different protein contexts may alter these recognition Properties, Dotentiallv influencing 3' splice sitea selection in the SF1/mBBP model system.