Genetic Analysis of RNA Binding Proteins

描述(申请人提供)：对RNA结合蛋白的兴趣(RNA-Bps) 近年来随着人们越来越多地意识到这些蛋白质 在多种细胞调节过程中起着关键作用 疾病。然而，支配RNA-BPS的许多基本原则 识别和组装它们的RNA靶标仍不清楚。这些 调查将集中在确定RNA-BPS的特征和他们的 对于它们的特异性和正确组装很重要的靶标。 我们将特别关注两种重要的RNA结合蛋白， HIV-1 Rev和果蝇Pumilio，每个都是一个大家庭的范例 RNA-bps。尽管还没有可用于REV的高分辨率结构， 最近对REV在其RNA靶标(RRE)上组装的遗传学研究 允许建立一个模型的三维结构 这种蛋白质的氨基末端的一半。该结构模型将由 使用生物物理、生化和突变策略的组合。 还需要研究的是REV结合对RRE几何结构的影响 以及影响早期阶段的这种分枝RNA结构的特征 多聚体版本总成。对Pumilio RNA结合域的研究将 包括RNA修饰、随机蛋白质突变和遗传筛选 确定对目标识别最关键的残基和 RNA结合专一性。用于浮标研究，一个广泛适用的 快速鉴定RNA结合蛋白突变体的遗传方法将是 精致，从而大大提高了速度和彻底性，其中复杂 突变文库可以进行筛选。 从这些研究中获得的知识应该是有价值的理解 基因调控和微生物致病机制，最终可能 在基于信使核糖核酸靶向的新型基因疗法的开发中具有一定的应用价值。

DESCRIPTION (provided by applicant):Interest in RNA-binding proteins (RNA-BPs) has burgeoned in recent years with the growing awareness that these proteins play a key role in a wide variety of cellular regulatory processes and in human disease. However, many of the fundamental principles that govern how RNA-BPs recognize and assemble on their RNA targets remain unclear. These investigations will focus on identifying the features of RNA-BPs and their targets that are important for their specificity and proper assembly. Particular attention will be devoted to two important RNA-binding proteins, HIV-1 Rev and Drosophila Pumilio, each a paradigm for a large family of RNA-BPs. Although no high-resolution structure is yet available for Rev, genetic studies of Rev assembly on its RNA target (the RRE) have recently allowed the formulation of a model for the three-dimensional structure of the amino-terminal half of this protein. This structural model will be tested by using a combination of biophysical, biochemical, and mutational strategies. Also to be investigated is the effect of Rev binding on the geometry of the RRE and the features of this branched RNA structure that influence the early stages of multimeric Rev assembly. Studies of the Pumilio RNA-binding domain will involve RNA modification and random protein mutagenesis and genetic screening to identify the residues that are most critical for target recognition and RNA-binding specificity. For use in the Pumilio studies, a broadly applicable genetic method for rapidly characterizing RNA-binding protein mutants will be refined so as to greatly enhance the speed and thoroughness with which complex mutant libraries can be screened. The knowledge derived from these studies should be of value for understanding mechanisms of gene regulation and microbial pathogenesis and could eventually be useful in the development of novel gene therapies based on mRNA targeting.