Northwestern techniques for the identification of RNA-binding proteins from cDNA expression libraries and the analysis of RNA-Protein interactions

Abstract

INTRODUCTION: This chapter discusses the critical role of RNA-binding proteins (RBPs) in various cellular processes, including gene expression, RNA processing, and viral replication. It emphasizes the necessity of identifying and characterizing these proteins to understand their functions in molecular biology. METHODOLOGY: The chapter outlines two primary protocols for identifying RBPs using Northwestern techniques. The first protocol involves producing recombinant proteins via bacteriophages and adsorbing them onto nitrocellulose membranes. The second protocol uses electroblotting to transfer proteins onto membranes. Both methods utilize 32P-labeled RNA probes to detect RNA-protein interactions. RESULTS: Northwestern techniques enable the efficient identification of cDNA clones expressing RBPs without the need for labor-intensive biochemical purification. Several RNA-protein interactions have been successfully identified and characterized using these methods, demonstrating their utility in detecting low-abundance RBPs in cDNA libraries and studying known RNA-protein interactions directly. CONCLUSIONS: Northwestern techniques provide a rapid, cost-effective tool for identifying and analyzing RBPs. These methods are particularly useful for studying RNA-protein interactions in biological systems where traditional genetic approaches are not well-developed. The described methodologies have proven effective in identifying proteins that interact with RNA and characterizing these interactions in various biological applications.