State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
ACS Chem Biol. 2021 Jun 18;16(6):1003-1010. doi: 10.1021/acschembio.0c00945. Epub 2021 May 19.
Imaging RNA-protein interaction in the cellular space with single molecule sensitivity is attractive for studying gene expression and regulation, but remains a challenge. In this study, we reported a photoactivatable trimolecular fluorescence complementation (TriFC) system based on fluorescent protein, mIrisFP, to identify and visualize RNA-protein interactions in living mammalian cells. We also combined this TriFC system with photoactivated localization microscopy (PALM), named the TriFC-PALM technique, which allowed us to image the RNA-protein interactions with single molecule sensitivity. Using this TriFC-PALM technique, we identified the actin-bundling protein, FSCN1, specifically interacting with the HOX Transcript Antisense RNA (HOTAIR). The TriFC-PALM imaging acquired a higher resolution compared with the traditional method of total internal reflection (TIRF) imaging. The TriFC-PALM thus provides a useful tool for imaging and identifying the RNA-protein interactions inside cells at the nanometer scale.
利用单分子灵敏度在细胞空间中成像 RNA-蛋白质相互作用对于研究基因表达和调控很有吸引力，但仍然具有挑战性。在这项研究中，我们报告了一种基于荧光蛋白 mIrisFP 的光激活三聚体荧光互补（TriFC）系统，用于在活的哺乳动物细胞中鉴定和可视化 RNA-蛋白质相互作用。我们还将这种 TriFC 系统与光激活定位显微镜（PALM）结合，命名为 TriFC-PALM 技术，该技术允许我们以单分子灵敏度对 RNA-蛋白质相互作用进行成像。使用这种 TriFC-PALM 技术，我们鉴定了肌动蛋白结合蛋白 FSCN1 与 HOX 转录反义 RNA（HOTAIR）的特异性相互作用。与传统的全内反射（TIRF）成像方法相比，TriFC-PALM 成像获得了更高的分辨率。因此，TriFC-PALM 为在纳米尺度上对细胞内的 RNA-蛋白质相互作用进行成像和鉴定提供了一种有用的工具。
