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Mapping the Transcriptome-Wide Landscape of RBP Binding Sites Using gPAR-CLIP-seq: Experimental Procedures
Ting Han1, John K Kim2,3
1Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX, 75390-9152, USA.
Methods in Molecular Biology (Clifton, N.J.)
|October 21, 2015
Summary
Global photoactivatable-ribonucleoside-enhanced cross-linking and precipitation followed by deep sequencing (gPAR-CLIP-seq) identifies all RNA-binding protein (RBP) binding sites in yeast. This method reveals global RNA-protein interactions and posttranscriptional gene regulation mechanisms.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- RNA-binding proteins (RBPs) regulate RNA fate, impacting gene expression.
- Understanding RBP-mediated regulation requires mapping their in vivo binding sites.
- Existing methods often focus on single RBPs, limiting global analysis.
Purpose of the Study:
- To describe the gPAR-CLIP-seq method for capturing and sequencing all RBP binding sites.
- To profile global RNA-protein interactions in vivo.
- To provide insights into posttranscriptional gene regulation.
Main Methods:
- Development and application of the gPAR-CLIP-seq technique.
- Utilizing photoactivatable ribonucleosides for cross-linking.
- Deep sequencing to identify bound RNA regions.
Main Results:
- gPAR-CLIP-seq successfully isolated and sequenced mRNA sites bound by the cellular "RBPome" in budding yeast.
- The method allows for comprehensive discovery of RBP binding sites.
- Generated a complete landscape of RBP binding sites.
Conclusions:
- gPAR-CLIP-seq is a powerful tool for profiling global RNA-protein interactions.
- The approach is applicable across various organisms and cell lines.
- Provides fundamental insights into posttranscriptional gene regulation and RNA cis-regulatory elements.