Genetic Analysis of RNA Binding Proteins

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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.