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Identification of circRNA-Interacting Proteins by Affinity Pulldown.
Jen-Hao Yang1, Poonam R Pandey2, Myriam Gorospe2
1Laboratory of Genetics and Genomics, Biomedical Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA. jen-hao.yang@nih.gov.
Methods in Molecular Biology (Clifton, N.J.)
|August 21, 2021
Summary
Researchers developed a new method to identify proteins bound to circular RNAs (circRNAs). This technique uses an antisense oligomer to pull down circRNA-protein complexes, aiding in understanding circRNA functions.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Circular RNAs (circRNAs) are a large class of RNA molecules formed by backsplicing.
- Despite their abundance, the functions of most circRNAs remain largely unknown.
- circRNA functions are hypothesized to involve interactions with proteins and other nucleic acids.
Purpose of the Study:
- To develop a robust method for identifying proteins that associate with specific circRNAs.
- To overcome limitations of existing methods that cannot distinguish circRNAs from linear counterparts.
Main Methods:
- An affinity pulldown assay using a biotinylated antisense oligomer targeting the circRNA-specific junction sequence.
- Streptavidin bead pulldown of circRNA-protein (circRNP) complexes.
- Mass spectrometry for protein identification, followed by validation using Western blot and other methods.
Main Results:
- The described protocol enables the specific isolation and identification of proteins associated with individual circRNAs.
- This method provides a critical tool for discovering circRNA-binding proteins.
Conclusions:
- The developed protocol is a flexible and robust approach for identifying circRNP components.
- This technique is essential for advancing the understanding of circRNA biological functions.
