A New Method for Studying RNA-binding Proteins on Specific RNAs

Weiping Sun; Ziheng Zhang; Ji-Long Liu; Min Zhuang

doi:10.21769/BioProtoc.4022

A New Method for Studying RNA-binding Proteins on Specific RNAs

Bio Protoc. 2021 May 20;11(10):e4022. doi: 10.21769/BioProtoc.4022.

Authors

Weiping Sun^{1

2

3}, Ziheng Zhang^{1

2

3}, Ji-Long Liu¹, Min Zhuang¹

Affiliations

¹ School of Life Science and Technology, ShanghaiTech University, Shanghai, China.
² University of Chinese Academy of Sciences, Beijing, China.
³ Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.

Abstract

Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT. Enzymatically deactivated Cas13a (dCas13a) is fused to the proximity labeling enzyme PafA. In the presence of a guide RNA, dCas13a binds specific target RNA region, while the fused PafA mediates the labeling of biotin-tagged Pup on proximal proteins. The labeled proteins can be enriched by streptavidin pull-down and identified by mass spectrometry. Here we describe the general procedure for capturing RNA-protein interactions using this method.

Keywords: CRISPR; CRUIS; PUP-IT; Proximity-based protein labeling; RNA-binding protein.