Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Conventional antibody-based CLIP assays face challenges with certain protein complexes, like Polycomb complexes.
• Efficient isolation of protein-RNA complexes is crucial for understanding gene regulation.

Purpose of the Study:

• To describe the denaturing cross-linked RNA immunoprecipitation (dCLIP) protocol for isolating protein-RNA complexes.
• To highlight the utility of dCLIP for studying challenging targets and identifying RNA binding motifs.

Main Methods:

• Biotin-tagging of target proteins.
• In vivo UV cross-linking of RNA to proteins.
• RNase protection assay.
• Isolation of denatured RNA-protein complexes using streptavidin affinity purification.

Main Results:

• Successful application of dCLIP to Polycomb complexes, overcoming limitations of conventional methods.
• Generation of small RNA footprints suitable for de novo motif analysis.
• Identification of RNA binding motifs associated with specific proteins.

Conclusions:

• The dCLIP protocol provides a robust method for isolating protein-RNA complexes, particularly for proteins that are difficult to study with traditional CLIP assays.
• dCLIP enables the identification of RNA binding motifs through de novo motif analysis of obtained RNA footprints.