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iCLIP for the thorough analysis of mRNA:protein interactions

Posted by Christoph Eckert on Aug 14, 2019 11:41:21 AM

Crystal structure of the anti-GFP VHH-Green Fluorescent Protein complex.
The GFP Nanobody is displayed blue and the GFP in green color.

UV crosslinking techniques are the method of choice for a comprehensive analysis of in-vivo-mRNA targets of an RNA-binding protein (RBP). In the recent publication of Olgeiser et al. (2019), the authors applied individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to study fungal mRNA transport. For this approach, they have used strains expressing GFP-tagged versions of the two RBPs Grp1 and Rrm4; an optimized protocol was developed to uncover that Grp1 and Rrm4 conjointly bind thousands of shared target messenger ribonucleoproteins (mRNPs) in the fungus U. maydis. The protein:RNA complexes were immunoprecipitated in a multiple detergent containing buffer using ChromoTek’s GFP-Trap Magnetic Agarose. This is a transcriptome‐wide view to an endosomal mRNA transport machinery.

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Topics: GFP, iCLIP

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