CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein

Abstract

Guilt by association remains a powerful argument for assigning function in biological processes: Proteins that bind a specific RNA are strongly implicated in modulating the fate and action of that RNA. Detecting such associations is a major goal in understanding RNA function. The cross-linking and immunoprecipitation (CLIP) method described here allows assignments of RNA-binding proteins to their RNA targets by using ultraviolet (UV) cross-linking to covalently capture close (near-covalent bond distances) association of RNA with protein, followed by immunopurification of the protein partner with extraction and subsequent characterization of the RNA partner sequence(s). Pools of target sequences can be captured and their genomic origins are studied so that regulatory and other functional relationships between the protein and its RNA targets can be inferred. The advantage of this protocol is that it captures only intimately associated RNAs and proteins, and so is expected to be highly specific. Some investigators report capturing cross-links between rRNA and nonribosomal proteins in cases where ribosomes are not efficiently cleared from the lysate before immunopurification of the target protein, so the limits of specificity for the method are yet to be fully explored. Its main disadvantages are its many steps and that it may fail to work for all proteins or capture all legitimate targets of any given protein if the necessary chemical groups are not optimally arranged for photocross-linking in the complex. However, properly performed, this method should provide many useful insights into the nature of the RNA ligands for an RNA-binding protein of interest.