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URL: https://dx.doi.org/DOI:10.17504/protocols.io.5ppg5mn

Authors: Rosey Bayne, Stefan Bresson, David Tollervey, Edward Wallace

Group: Wallace lab for Fungal RNA

Summary: CRAC (Cross-linking and cDNA Analysis) is a method that uses UV-crosslinking to identify which RNAs bind to an RNA-Binding Protein of interest and the specific sites in the RNA where this binding occurs. CRAC is one of the family of UV crosslinking and immunoprecipitation (CLIP) protocols, reviewed by Lee and Ule (DOI: 0.1016/j.molcel.2018.01.005).The protein is tagged with with a tandem affinity tag: in this protocol we use an HTP tag (His6-TEV-ProteinA) that allows immuno-affinity purification of the protein with its cross-linked RNAs by binding of the ProteinA moiety of the tag to IgG Sepharose columns. After washing, the protein is then released from the column by cleavage with TEV protease and subjected to nickel-affinity purification through the His6 moiety under highly denaturing conditions. This allows accurate mapping of RNA binding sites (Bohnsack, et al, DOI: 10.1016/B978-0-12-396546-2.00013-9) ; Granneman, S. et al (DOI: 10.1073/pnas.0901997106 ). The protocol can also be adapted for use with HTF (His6-TEV-FLAG3) tags (Tree, J.J. et al,  DOI: 10.1016/j.molcel.2014.05.006) or HF (His8-Ala4-FLAG) tags (Bresson, S., Shchepachev, V., Spanos, C., Turowski, T., Rappsilber, J. and Tollervey, D.(2020): DOI: /10.1101/2020.05.14.096354) by replacing the IgG sepharose column with anti-FLAG magnetic beads and elution with excess FLAG peptide, removing the need for TEV cleavage which can be inefficient. The cDNA libraries generated allow a transcriptome -wide analysis of the interactome of the protein.

Affiliations: School of Biological Sciences, University of Edinburgh, Wellcome Centre for Cell Biology, University of Edinburgh, Wellcome Centre for Cell Biology, University of Edinburgh, School of Biological Sciences, University of Edinburgh

Version: 1

Publication Date: 2020

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