Journal Article

A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins

Zavolan, Mihaela, Khorshid, Mohsen, Kishore, Shivendra, Hausser, Jean, Jaskiewicz, Lukasz, Burger, Lukas

2011

Overview

The comparison of cross-linking and immunoprecipitation (CLIP) and photoactivatable ribonucleoside–enhanced CLIP (PAR-CLIP) protocols shows specific biases of each method in enriching subsets of binding sites of RNA-binding proteins and shows ways around these biases. 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.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: 631/1647/2217 / 631/1647/48 / 631/45/612/1230 / Analysis / Binding proteins / Binding Sites / Bioinformatics / Biological Microscopy / Biological Techniques / Biomedical and Life Sciences / Biomedical Engineering/Biotechnology / Chemical properties / Cross-Linking Reagents - chemistry / Genetic aspects / Immunoenzyme technique / Immunoprecipitation - methods / Life Sciences / Methods / Molecular biology / Mutation / Physiological aspects / Protein binding / Proteins / Proteomics / Ribonucleic acid / RNA / RNA-Binding Proteins - analysis / RNA-Binding Proteins - chemistry / RNA-Binding Proteins - metabolism / Scientific method