Shengcheng Deng
Tianyu Ge
Miaoman Ye
Jianping Yu
Song Lin
Wenbin Ma
Zhou Songyang
1 Sun Yat-sen Memorial Hospital, Sun Yat-sen University;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;
2 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China;
3 Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
Funds: We would like to thank Dr. Dan Liu for helping with the manuscript. This work was supported by the National Key Research and Development Program of China (2017YFA0102801, 2018YFA0107003), National Natural Science Foundation of China (91640119, 91749113, 31570827, 31871479 and 31930058), Natural Science Foundation of Guangdong Province (2017A030313116) and the China Postdoctoral Science Foundation (2018M631021).
Received Date: 2019-11-20
Rev Recd Date:2020-02-19
Abstract
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.Keywords: CRISPR/Cas9 genome editing,
endogenous lncRNA labeling,
MS2-MCP,
NEAT1,
paraspeckle dynamics
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