Individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to determine protein–RNA interactions

Abstract

RNA-binding proteins (RBPs) interact with and determine the fate of many cellular RNA transcripts. In doing so they help direct many essential roles in cellular physiology, while their perturbed activity can contribute to disease etiology. In this chapter we detail a functional genomics approach, termed individual nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), that can determine the interactions of RBPs with their RNA targets in high throughput and at nucleotide resolution. iCLIP achieves this by exploiting UV-induced covalent cross-links formed between RBPs and their target RNAs to both purify the RBP–RNA complexes under stringent conditions, and to cause reverse transcription stalling that then identifies the direct cross-link sites in the high throughput sequenced cDNA libraries.