Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in
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Protein & Cell
RESEARCH ARTICLE Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/ Cas9-mediated knock-in Bohong Chen1, Shengcheng Deng1, Tianyu Ge1, Miaoman Ye1, Jianping Yu1, Song Lin1, Wenbin Ma1, Zhou Songyang1,2,3& 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 & Correspondence: [email protected] (Z. Songyang) Received November 20, 2019 Accepted February 19, 2020
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
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13238-020-00706-w) contains supplementary material, which is available to authorized users.
© The Author(s) 2020
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 INTRODUCTION Noncoding RNAs (ncRNAs) have been characterized as the dark matter of the human genome because they do not encode protein sequences despite taking up a major portion of our transcriptome (Derrien et al., 2012; Evans et al., 2016; Jandura and Krause, 2017). ncRNAs that longer than 200 nt are called long noncoding RNAs (lncRNAs) and are particularly interesting due to their important roles in a variety of cellular activities (Marchese et al., 2017; Kopp and Mendell, 2018; Yao et al., 2019). It is well documented that lncRNAs can fold into stru
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