CBRPP: a new RNA-centric method to study RNA–protein interactions

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 <i>in vitro</i>. 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.

RNA与蛋白质是两类相互关联的生物分子，可通过物理相互作用调控彼此的生命周期与功能。异常的RNA-蛋白质相互作用会引发细胞功能失调与人类疾病。因此，绘制RNA-蛋白质相互作用网络，对于理解细胞过程及相关疾病的发病机制至关重要。目前已有多种实用的以蛋白质为中心的RNA-蛋白质相互作用研究方法被报道，但具备稳健性能的以RNA为中心的研究方法却为数不多。本研究开发了基于CRISPR的RNA邻近蛋白质组学（CRISPR-based RNA proximity proteomics, CBRPP），这是一种全新的以RNA为中心的研究方法，可在天然细胞环境中，无需对靶标RNA进行预编辑、体外交联，亦无需在体外对RNA-蛋白质复合物进行操作的前提下，鉴定与目标内源RNA相关的蛋白质。CBRPP基于dCas13与邻近标记酶（proximity-based labelling, PBL）的融合蛋白构建而成。dCas13可将PBL酶高特异性地递送至靶标RNA，而PBL酶则会对靶标RNA周围的蛋白质进行标记，随后通过质谱技术对这些蛋白质进行鉴定。