The Development and Application of a Method to Quantitatively Identify RNA Binding Sites, and Whole Transcript Targets of RNA Binding Proteins

Abstract

RNA binding proteins (RBPs) and non-coding RNAs orchestrate gene expression in part through the recognition specific sites in mRNA. Thus understanding the connection between binding to specific sites and regulation of the whole transcript is essential. Current methods to do this can either identify the binding sites or quantitate binding to whole transcripts, but not both. Furthermore reliance of binding site detection on ultraviolet crosslinking results in inefficient identification of binding sites, and insufficient data to assess binding strength at sites. I have overcome these limitations by combining aspects of current methods to develop a new method called DO-RIP-seq (digestion optimization RNA immunoprecipitations with deep sequencing) that can quantitate the binding strength of RBPs at sites in mRNA, and also relate binding sites to binding of the whole mRNA. DO-RIP-seq was developed using the well-studied RBP ELAVL1/HuR as a test case, and applied to the less well-studied RBP known as RBM38/RNPC1. The quantitative data from DO-RIP-seq out-performed current binding site methods at predicting other features of the binding sites of HuR and RBM38, for example the lack of RNA secondary structure, and preferences in binding to particular sub-motifs. My studies indicate that DO-RIP-seq will be useful in uncovering the determinants of RNA-protein interactions, and studying dynamic biological processes that could modulate these interactions.