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Updated: May 21, 2026
PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins
Published on: July 2, 2010
Identification of RNA helicase target sites by UV cross-linking and analysis of cDNA
Markus T Bohnsack1, David Tollervey, Sander Granneman
1Cluster of Excellence Macromolecular Complexes, Institute for Molecular Biosciences, Goethe University Frankfurt, Frankfurt, Germany.
Abstract:
Many RNA helicases have been implicated in one or more pathways of RNA metabolism, but only in a very few cases have their target sites on the RNA been identified. Here, we give a detailed description of the UV cross-linking and analysis of cDNA (CRAC) method, and its application to the identification of binding sites of RNA-interacting helicases. CRAC makes use of a bipartite tag on the protein of interest and includes a purification step under highly denaturing conditions. This is particularly important for the accurate mapping of binding sites within large RNA-protein complexes--such as spliceosomes or preribosomes. Partial RNase digestion leaves a footprint of the protein covering the interaction site, and the UV cross-linking sites are frequently highlighted by microdeletions in cDNA sequence reads. Deep sequencing of cDNA libraries generated from cross-linked RNA fragments allows a genome-wide analysis of the interactome of RNA-binding proteins. In the case of RNA helicases, this has proven to be an important step toward their functional analysis.
Insights
The UV cross-linking and analysis of cDNA (CRAC) method accurately maps RNA helicase binding sites. This technique aids in understanding RNA helicase function by identifying their specific targets on RNA molecules.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- RNA helicases play crucial roles in RNA metabolism pathways.
- Identifying specific RNA binding sites for helicases has been challenging.
- Understanding these interactions is key to elucidating helicase function.
Purpose of the Study:
- To provide a detailed description of the UV cross-linking and analysis of cDNA (CRAC) method.
- To demonstrate the application of CRAC for identifying RNA helicase binding sites.
- To enable accurate mapping of protein-RNA interactions within large complexes.
Main Methods:
- Utilized a bipartite tag on the protein of interest for purification.
- Employed highly denaturing conditions during purification to maintain complex integrity.
- Performed partial RNase digestion to generate protein footprints and UV cross-linking analysis of cDNA (CRAC) for deep sequencing of RNA fragments.
Main Results:
- CRAC successfully identified specific RNA binding sites for RNA helicases.
- The method accurately maps binding sites within large RNA-protein complexes like spliceosomes and preribosomes.
- Microdeletions in cDNA sequence reads frequently highlight UV cross-linking sites.
Conclusions:
- The CRAC method is effective for genome-wide analysis of RNA-binding protein interactomes.
- CRAC provides crucial insights into the functional roles of RNA helicases by identifying their RNA targets.
- This technique advances the functional characterization of RNA helicases.
