Individual Nucleotide Resolution UV Cross-Linking and Immunoprecipitation (iCLIP) to Determine Protein–RNA Interactions
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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.
Key wordsiCLIP CLIP RNA-binding protein RNA Protein–RNA interactions Post-transcriptional regulation
The iCLIP protocol described is based on previous versions developed in the Ule and Konig labs by many individuals. I would like to extend thanks to all those who have contributed to the method development over the years. I would also like to thank Prof. Jernej Ule for critical reading of the chapter, and Prof. Jernej Ule and Flora Lee for providing the adapter oligo used in this protocol. This work is supported by an Edmond Lily Safra Fellowship to C.R.S.
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