Transcriptome-wide identification of in vivo interactions between RNAs and RNA-binding proteins by RIP and PAR- CLIP assays

Abstract

Comprehensive genomic and computational studies in the era of high-throughput sequencing revealed that the major proportion of the human genome is transcribed. This novel insight confronted the scientific community with new questions concerning the expanded role of RNA, especially noncoding RNA (ncRNA), in cellular pathways. In recent years, there has been mounting evidence that ncRNAs and RNA binding proteins (RBPs) are involved in a wide range of biological processes, such as developmental transitions, cell differentiation, stress response, genome organization, and regulation of gene expression. In particular, in the chromatin field long noncoding RNAs (lncRNAs) have drawn increasing attention to their function in epigenetic regulation due to the fact that they were found to interact with multiple chromatin regulators and modifiers. Recently, techniques to study the extent of RNA-protein interactions have been developed in many research laboratories. Here we describe protocols for RNA Immunoprecipitation- Sequencing (RIP-Seq) and Photoactivatable-Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation combined with deep sequencing (PAR-CLIP-Seq) to identify RNA targets of RNAbinding proteins (RBPs) on a transcriptome-wide level, discussing advantages and drawbacks.