RNA molecules function as messengers or noncoding adaptor molecules, structural components, and regulators of genome organization and gene expression. Their roles and regulation are mediated by other molecules they interact with, especially RNA binding proteins (RBPs). Here we report RNA proximity labeling (RPL), an RNA-centric method based on fusion of an endonuclease-deficient Type VI CRISPR-Cas protein (dCas13b) and engineered ascorbate peroxidase (APEX2) to discover in vivo target RNA proximal proteins (RPPs) through proximity-based biotinylation. U1 RPPs enriched by proximity-based biotinylation included both U1 snRNA canonical and noncanonical functions-related proteins. In addition, profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs for poly(A) tails and also provided novel evidence for poly(A)+ RNA 5 prime-3 prime proximity and expanded subcellular localizations. Our results suggest that RPL is a rapid approach for identifying both interacting and neighboring proteins associated with target RNA molecules in their native cellular contexts.

中文翻译：

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通过邻近依赖性生物素化在体内发现RNA近端蛋白质

RNA分子充当信使或非编码衔接子分子，结构组件以及基因组组织和基因表达的调节剂。它们的作用和调节由与其相互作用的其他分子介导，尤其是RNA结合蛋白（RBP）。在这里，我们报告了RNA邻近标记（RPL），这是一种以RNA为中心的方法，基于融合内切核酸酶的VI型CRISPR-Cas蛋白（dCas13b）和工程化的抗坏血酸过氧化物酶（APEX2），以发现体内靶RNA近端蛋白（RPP）通过基于邻近的生物素化。通过基于邻近的生物素化作用富集的U1 RPP包括U1 snRNA规范和非规范功能相关蛋白。此外，聚（A）尾部近端蛋白的分析发现了聚（A）尾部的RBP的预期类别，也为聚（A）+ RNA 5 prime-3引物邻近和扩展的亚细胞定位提供了新的证据。我们的结果表明，RPL是一种在目标细胞天然分子中识别与目标RNA分子相关的相互作用蛋白和邻近蛋白的快速方法。