Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Experimental Procedures

Abstract

An estimated 5–10 % of protein-coding genes in eukaryotic genomes encode RNA-binding proteins (RBPs). Through dynamic changes in RNA recognition, RBPs posttranscriptionally regulate the biogenesis, transport, inheritance, storage, and degradation of RNAs. Understanding such widespread RBP-mediated posttranscriptional regulatory mechanisms requires comprehensive discovery of the in vivo binding sites of RBPs. Here, we describe the experimental procedures of the gPAR-CLIP-seq (global photoactivatable-ribonucleoside-enhanced cross-linking and precipitation followed by deep sequencing) approach we recently developed for capturing and sequencing regions of the transcriptome bound by RBPs in budding yeast. Unlike the standard PAR-CLIP method, which identifies the bound RNA substrates for a single RBP, the gPAR-CLIP-seq method was developed to isolate and sequence all mRNA sites bound by the cellular “RBPome.” The gPAR-CLIP-seq approach is readily applicable to a variety of organisms and cell lines to profile global RNA–protein interactions underlying posttranscriptional gene regulation. The complete landscape of RBP binding sites provides insights to the function of all RNA cis-regulatory elements in an organism and reveals fundamental mechanisms of posttranscriptional gene regulation.