CBRPP: a new RNA-centric method to study RNA–protein interactions

ABSTRACT

RNA and protein are interconnected biomolecules that can influence each other’s life cycles and functions through physical interactions. Abnormal RNA–protein interactions lead to cell dysfunctions and human diseases. Therefore, mapping networks of RNA–protein interactions is crucial for understanding cellular processes and pathogenesis of related diseases. Different practical protein-centric methods for studying RNA–protein interactions have been reported, but few robust RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with an endogenous RNA of interest in native cellular context without pre-editing of the target RNA, cross-linking or RNA–protein complexes manipulation in vitro. CBRPP is based on a fusion of dCas13 and proximity-based labelling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.

KEYWORDS:

• RNA-protein interactions
• dRfxCas13d
• dPspCas13b
• bioid2
• turboid
• apex2
• basu

Supplementary material

Supplemental data for this article can be accessed here.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31570891) and the National Key Research and Development Program of China (Grant #2016YFA0500302).

Author Contributions

Y.L. and F.Y. conceived this project. Y.L. analyzed the data and wrote the paper. Y.L., SD.L., L.C., H.D. and F.Y. revised the paper. Y.L., SD.L., L.C. and YJ.L. performed most experiments. SJ.L. contributed to imaging.

Disclosure statement

The authors declare no competing interests.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [31570891]; National Key Research and Development Program of China [2016YFA0500302].