9] In vitro genetic analysis of RNA-binding proteins using phage display libraries

Abstract

Publisher Summary This chapter is divided into two main sections. The first describes the various aspects of displaying RNA- binding proteins (BPs) on filamentous phage: assessing and optimizing export, the production and purification of fusion phage, the preparation of the RNA target, and the selective binding of fusion phage. The second section describes how to use phage display for the in vitro genetic analysis of RNA-BPs: the synthesis of a combinatorial phagemid library, the production of the corresponding fusion phage library, and the genetic selection of RNA-BP variants, with certain binding properties. In both sections, the studies of the U1A protein are used to illustrate these methods. The U1A protein is a component of the U1 small nuclear ribonucleo-protein particle that forms a part of the spliceosome. It is a representative of the largest family of RNA-BPs that binds RNA through an RNA recognition motif (RRM) domain. The various RRM domains of this protein family enable the different family members to bind to a wide variety of RNA sequences and structures, with a high degree of specificity. The U1A protein contains two RRM domains, the N-terminal of which is responsible for binding the protein to hairpin II of U1 RNA (UlhpII). This chapter describes the display of the U1A N-terminal RRM domain on phage, the construction of a combinatorial library of fusion phage displaying U1A mutants, and the selection of U1A variants that bind tightly to U1hpII RNA.