The Mechanism of RNA Binding by the U1A Protein: A Model for Understanding the Kinetics of RNA/Protein Interactions

University Of Southern California, Los Angeles CA

Investigators

Abstract

RNA-binding proteins are critically important to many regulatory processes in the cell. The largest family of RNA-binding proteins carries one of more RNA recognition motif domains (RRMs). RRM-containing proteins occur in all living organisms, and play a role in most processes involving RNA, including splicing, RNA export, translation, and mRNA decay. U1A is a spliceosomal protein that carries two RRMs. Its N-terminal RRM mediates tight and specific binding to its RNA hairpin target (U1hpII)in U1 small nuclear RNA. U1A is one of the most extensively studied RNA-binding proteins and has been used as a paradigm for specific and tight RNA recognition by RRM-type proteins. However, to date the knowledge of the mechanism of RNA-binding by U1A and other RNA-binding proteins has been largely limited to a static image, in which the dynamics underlying the interactions are poorly understood. This project is aimed at obtaining detailed kinetic insight into the U1A/U1hpII interaction using powerful state- of-the-art BIACORE technology to observe, in real time, the assembly and dissociation of the RNA/protein complex. Analysis of the kinetics of the U1A/U1hpII interaction will help elucidate the sequence of events that occurs during binding. More importantly, it will also provide general insights into the dynamics of RNA/protein interactions, an area that has thus far remained largely unexplored. Knowledge of the kinetics of RNA/protein interactions is highly relevant, since biological systems are dynamic; the rates of binding and release can affect the sequential assembly of RNA/protein complexes and the function and intracellular localization of RNA- binding proteins, as well as their ability to compete for binding sites on RNA.