基于CRISPR的活细胞RNA结合蛋白图谱分析

CRISPR-based RNA-binding protein mapping in live cells.

作者信息

Chen Baiwen, Shi Haiyan, Zhang Jia, Zhou Chun, Han Miaomiao, Jiang Wenxiu, Lai Yuting, Tu Xia, Li Huabin

机构信息

ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital,Fudan University, Shanghai, 200031, China.

Department of ER, CHINA RESOURCES & WISCO GENERAL HOSPITAL, Wuhan University of Science and Technology, No.209 Metallurgical Avenue, Qingshan District, Wuhan, Hubei, 430080, China.

出版信息

Biochem Biophys Res Commun. 2021 Oct 29;583:79-85. doi: 10.1016/j.bbrc.2021.10.059.

DOI:10.1016/j.bbrc.2021.10.059

PMID:34735883

Abstract

RNA-binding proteins (RBPs) are involved in all aspects of RNA metabolism, and RNA-RBP interactions are important for cell homeostasis and viral replication. The global RNA-binding proteome was recently reported; however, little is known about the proteins that bind to specific RNAs. In this study, we describe a novel CRISPR-based RNA interaction proteomics method in live cells. In brief, dCas13a with an HA tag was expressed in cells and bound to an RNA of interest with the help of gRNA. The RNA-protein complexes physically bound to dCas13a-HA were crosslinked using UV light and captured using anti-HA beads, after which the proteins were purified and identified using mass spectrometry. We optimized this system and subsequently applied it to U1 small nuclear RNA, which revealed 226 proteins.

摘要

RNA结合蛋白（RBPs）参与RNA代谢的各个方面，RNA与RBP的相互作用对于细胞稳态和病毒复制至关重要。最近报道了全球RNA结合蛋白质组；然而，对于与特定RNA结合的蛋白质却知之甚少。在本研究中，我们描述了一种在活细胞中基于CRISPR的新型RNA相互作用蛋白质组学方法。简而言之，带有HA标签的dCas13a在细胞中表达，并在gRNA的帮助下与感兴趣的RNA结合。与dCas13a-HA物理结合的RNA-蛋白质复合物用紫外线交联，并用抗HA珠子捕获，然后纯化蛋白质并使用质谱进行鉴定。我们优化了该系统，随后将其应用于U1小核RNA，结果显示有226种蛋白质。