title: A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins.
abstract: Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.
authors.affiliation: Biozentrum, University of Basel and Swiss Institute of Bioinformatics, Basel, Switzerland.
authors.forename: Shivendra
authors.initials: S
authors.lastname: Kishore
authors.forename: Lukasz
authors.initials: L
authors.lastname: Jaskiewicz
authors.forename: Lukas
authors.initials: L
authors.lastname: Burger
authors.forename: Jean
authors.initials: J
authors.lastname: Hausser
authors.forename: Mohsen
authors.initials: M
authors.lastname: Khorshid
authors.forename: Mihaela
authors.initials: M
authors.lastname: Zavolan
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D047468:Immunoprecipitation / Q000379:methods*; D016601:RNA-Binding Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D016601:RNA-Binding Proteins