Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in
Live cell imaging and proteomic profiling of endogenous NEAT1 lncRNA by CRISPR/Cas9-mediated knock-in
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通过 CRISPR/Cas9 介导的基因敲入对内源性 NEAT1 lncRNA 进行活细胞成像和蛋白质组分析
DOI：
10.1007/s13238-020-00706-w
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发表时间：
2020-05-26
期刊：
影响因子：
21.1
通讯作者：
Zhou Songyang
中科院分区：
文献类型：
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作者：
Chen, Bohong;Deng, Shengcheng;Zhou Songyang
在哺乳动物细胞中，长链非编码RNA（lncRNA）与蛋白质形成复合物以执行各种生物学功能，例如基因转录、RNA加工和其他信号传导活动。然而，在活细胞中追踪内源性lncRNA动力学和筛选lncRNA相互作用蛋白的方法是有限的。在这里，我们报告了CERTIS（CRISPR介导的内源性lncRNA跟踪和免疫沉淀系统）的开发，通过CRISPR/Cas9技术将24个重复的MS 2标签精确插入lncRNA基因座的远端来可视化和分离内源性lncRNA。在这项研究中，我们表明CERTIS有效地标记paraspeckle lncRNA NEAT 1，而不干扰其生理特性，并可以监测NEAT 1的内源性表达变化。此外，CERTIS在活细胞中NEAT 1动力学的短期和长期跟踪方面均显示出优越的上级性能。我们发现NEAT 1和paraspeckles对拓扑异构酶I特异性抑制剂敏感。此外，MS 2标记的NEAT 1 lncRNA的RNA免疫沉淀（RIP）成功地揭示了paraspeckle的几个新的蛋白质组分。我们的研究结果支持CERTIS作为一种工具，适合于跟踪空间和时间的lncRNA调节活细胞，以及研究lncRNA-蛋白质相互作用。
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.