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.

• PMID: 26483017
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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't

Authors

Han T, Kim JK

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