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Abstract：
RNA and protein are interconnected biomolecules that can influence each other\'s life cycles and functions through physical interactions. Abnormal RNA-protein interactions lead to cell dysfunctions and human diseases. Therefore, mapping networks of RNA-protein interactions is crucial for understanding cellular processes and pathogenesis of related diseases. Different practical protein-centric methods for studying RNA-protein interactions have been reported, but few robust RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with an endogenous RNA of interest in native cellular context without pre-editing of the target RNA, cross-linking or RNA-protein complexes manipulation in vitro. CBRPP is based on a fusion of dCas13 and proximity-based labelling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.
摘要：
RNA和蛋白质是相互关联的生物分子，可以通过物理相互作用影响彼此的生命周期和功能。异常的RNA-蛋白质相互作用导致细胞功能障碍和人类疾病。因此,RNA-蛋白质相互作用的映射网络对于理解相关疾病的细胞过程和发病机制至关重要。已经报道了研究RNA-蛋白质相互作用的不同实用的以蛋白质为中心的方法，但是很少有强大的以RNA为中心的方法存在。这里,我们开发了基于CRISPR的RNA邻近蛋白质组学（CBRPP），一种新的以RNA为中心的方法，用于在天然细胞环境中鉴定与感兴趣的内源性RNA相关的蛋白质，而无需预先编辑靶RNA，在体外操作交联或RNA-蛋白质复合物。CBRPP基于dCas13和基于邻近的标记(PBL)酶的融合。dCas13可以将PBL酶递送到靶RNA，具有高特异性，而PBL酶标记靶RNA的周围蛋白质，然后通过质谱鉴定。
