Chapter title |
Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Experimental Procedures.
|
---|---|
Chapter number | 5 |
Book title |
Yeast Functional Genomics
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Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3079-1_5 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3078-4, 978-1-4939-3079-1
|
Authors |
Han, Ting, Kim, John K, Ting Han, John K. Kim |
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. |
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