超高分辨成像技术研究细胞内长链非编码RNA-HOTAIR与蛋白质相互作用

长链非编码RNA(lncRNA)是一种转录本长度超过200个核苷酸的RNA,具有复杂的生物学功能.HOTAIR (HOX antisense intergenic RNA)是一种与多种肿瘤的发生发展和转移有关的lncRNA. HOTAIR可以通过与关键蛋白发生相互作用而行使功能，原位、精细、可视的解析HOTAIR-蛋白质相互作用能更好地理解其分子作用机制。然而目前lncRNA-蛋白质相互作用的可视化研究仍缺乏有效的方法和手段。本申请拟结合蛋白片段互补技术和光控mIrisFP荧光蛋白的性能，拆分构建mIrisFP三分子荧光互补系统，建立用于lncRNA-蛋白质相互作用细胞内STORM/PALM超高分辨成像分析的新方法,进一步通过在纳米尺度可视化分析细胞内HOTAIR与蛋白质的相互作用，明确HOTAIR与其相互作用蛋白精确的亚细胞定位以及空间相互作用关系,阐明其相互作用的分子机制.

Numerous long non-coding RNAs (lncRNAs) were found to play significant roles in cancer progression. LncRNA HOTAIR (HOX antisense intergenic RNA) is dys-regulated in various kinds of cancer cells, and HOTAIR expression level in primary tumours is a powerful predictor of eventual metastasis and death. HOTAIR is highly structured, it can bridges together two different chromatin modifying complexes to.target genes to coordinately alter the chromatin state and enforce gene silencing. A full understanding of HOTAIR-protein interactions during the cancer invasion and metastasis is essential but still far beyond completion. In this proposal, based on the technique of protein-fragment complementation and fluorescent protein mIrisFP with photoactivatable and photoswitchable fluorophore, we will attempt to.build a new fluorescent mIrisFP-based trimolecular fluorescence complementation (TriFC) system for STORM/PALM super-resolution fluorescence technique for imaging HOTAIR-protein interactions. Then, the newly constructed mIrisFP-TriFC system will be used for super-resolution fluorescence imaging lncRNA-protein interactions, and shed light on understanding and predicting lncRNA-protein interactions in cells.