Chapter 6 - Rapid Identification and Cloning of Sequence-Specific RNA Binding Proteins

Abstract

This chapter presents a UV cross-linking approach to identify sequence-specific Ribonucleic acid (RNA) binding proteins in in vitro reconstitution assays. A typical result in the early stages in studies of messenger ribonucleic acid (mRNA) biology is the identification of a region of a transcript that plays a pivotal role in the control of a posttranscriptional process. The next key focus of the study is determining the mechanism by which this RNA signal functions. It is likely that many RNA signals work through the sequence- or structure-specific assembly of protein–RNA complexes. This chapter provides an overview of approaches used to identify the protein binding site on the transcript. These methodologies provide a reasonably rapid and efficient strategy for the identification and molecular characterization of RNA binding proteins. The chapter discusses in detail about the preparation of extracts and preparation of RNAs. Analysis of UV cross-linked protein-RNA complexes, identification of protein–RNA complexes, and determination of RNA sequence requirements for protein binding is also discussed in detail. The chapter also highlights cloning complementary Deoxyribonucleic acids (cDNAs) for RNA binding proteins by northwestern screening.