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.
Highlights
Noncoding RNAs 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 and are interesting due to their important roles in a variety of cellular activities (Marchese et al, 2017; Kopp and Mendell, 2018; Yao et al, 2019)
We show that CRISPR-mediated endogenous long noncoding RNA (lncRNA) tracking and immunoprecipitation system (CERTIS) effectively labeled endogenous paraspeckle lncRNA nuclear-enriched abundant transcript 1 (NEAT1) without disturbing its physiologic properties and could be used to quantitatively monitor the expression variation of endogenous NEAT1
EM and super-resolution microscopy have been used to reveal that paraspeckles are composed of fused chains of spherical subcompartments, with NEAT1_2 arranged perpendicular to the longer axis of paraspeckles and its 5′ and 3′ ends facing outward (Hu et al, 2016; West et al, 2016)
Summary
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 interesting due to their important roles in a variety of cellular activities (Marchese et al, 2017; Kopp and Mendell, 2018; Yao et al, 2019). No distinct catalytic activity has been found to occur within paraspeckles, while it is commonly believed that the molecular basis of paraspeckle function mainly consequent on the sequestration of certain RNA species and paraspeckle protein components by the scaffold of NEAT1 lncRNA (Chen and Carmichael, 2009; Hirose et al, 2014; Wang et al, 2018) Study of such a highly dynamical lncRNA has been limited by a lack of efficient and versatile method that can both track endogenous lncRNA dynamics in live cells and identify lncRNA interacting proteins. Our results suggest that CERTIS is a tool suitable to track both spatial and temporal lncRNA dynamics in live cells as well as study the lncRNA-protein interactomes
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