Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in.
10.1007/s13238-020-00706-w
- Author:
Bohong CHEN
1
,
2
;
Shengcheng DENG
1
,
2
;
Tianyu GE
1
,
2
;
Miaoman YE
1
,
2
;
Jianping YU
1
,
2
;
Song LIN
1
,
2
;
Wenbin MA
1
,
2
;
Zhou SONGYANG
1
,
3
Author Information
1. Sun Yat-sen Memorial Hospital, Sun Yat-sen University
2. MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China.
3. MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research, SYSU-BCM Joint Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275, China. songyanz@mail.sysu.edu.cn.
- Publication Type:Journal Article
- Keywords:
CRISPR/Cas9 genome editing;
MS2-MCP;
NEAT1;
endogenous lncRNA labeling;
paraspeckle dynamics
- From:
Protein & Cell
2020;11(9):641-660
- CountryChina
- Language:English
-
Abstract:
In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.
