Genetic Analysis of RNA Binding Proteins

描述（由申请人提供）：对RNA结合蛋白（RNA-BP）的兴趣 近年来，随着人们越来越意识到这些蛋白质 在多种细胞调节过程和人体内发挥关键作用， 疾病然而，许多控制RNA-BPs的基本原则 识别和组装它们的RNA目标仍然不清楚。这些 研究将集中在确定RNA-BPs的特征及其 对于它们的特异性和正确组装很重要的靶点。 特别注意将致力于两个重要的RNA结合蛋白， HIV-1 Rev和果蝇Pumilio，每一个都是一个大家族的典范。 RNA-BP。虽然还没有高分辨率的结构可用于Rev， 最近，Rev在其RNA靶标（RRE）上组装的遗传学研究 允许制定一个模型的三维结构的 氨基末端的一半该结构模型将由 使用生物物理、生物化学和突变策略的组合。 还将研究Rev结合对RRE几何形状的影响 以及这种分支RNA结构的特征， 多聚体Rev组装。Pumilio RNA结合域的研究将 涉及RNA修饰和随机蛋白质诱变以及遗传筛选 以鉴定对靶标识别最关键的残基， RNA结合特异性。为了在Pumilio研究中使用， 一种快速表征RNA结合蛋白突变体的遗传学方法， 完善，以大大提高速度和彻底性， 可以筛选突变体文库。 从这些研究中获得的知识应该对理解有价值 基因调控和微生物致病机制，并最终可能 可用于开发基于mRNA靶向的新型基因疗法。

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.