【摘 要】
：
In mammalian cells,long noncoding RNAs (IncRNAs)form complexes with proteins to execute various biological functions such as gene transcription,RNA processing a
【机 构】
：
Sun Yat-sen Memorial Hospital, Sun Yat-sen University;MOE Key Laboratory of Gene Function and Regula
论文部分内容阅读
In mammalian cells,long noncoding RNAs (IncRNAs)form complexes with proteins to execute various biological functions such as gene transcription,RNA processing and other signaling activities.However,methods to track endogenous IncRNA dynamics in live cells and screen for IncRNA interacting proteins are limited.Here,we report the development of CERTIS (CRISPR-mediated Endogenous IncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous IncRNA,by precisely inserting a 24-repeat MS2 tag into the distal end of IncRNA locus through the CRISPR/Cas9 technology.In this study,we show that CERTIS effectively labeled the paraspeckle IncRNA 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 IncRNA successfully revealed several new protein components of paraspeckle.Our results support CERTIS as a tool suitable to track both spatial and temporal IncRNA regulation in live cells as well as study the IncRNA-protein interactomes.
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