Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Experimental Procedures.
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.
Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX, 75390-9152, USA.
Ting
T
Han
Life Sciences Institute, University of Michigan, Ann Arbor, MI, 48109-2216, USA. jnkim@umich.edu.|Department of Biology, Johns Hopkins University, Baltimore, MD, 21211, USA. jnkim@umich.edu.
John K
JK
Kim
D001665:Binding Sites / Q000235:genetics; D005057:Eukaryotic Cells; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D016678:Genome; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D059467:Transcriptome / Q000235:genetics*
D016601:RNA-Binding Proteins
4-Thiouracil; Posttranscriptional gene regulation; RNA binding protein s; UV cross-linking