In-cell identification and measurement of RNA-protein interactions
In-cell identification and measurement of RNA-protein interactions
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DOI：
10.1038/s41467-019-13235-w
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发表时间：
2019-11-22
影响因子：
16.6
通讯作者：
Shkumatava, Alena
中科院分区：
文献类型：
--
作者：
Graindorge, Antoine;Pinheiro, Ines;Shkumatava, Alena
调节RNA通过RNA结合蛋白（RBP）发挥其细胞功能。因此，确定RNA-蛋白质相互作用是从分子水平理解调控RNA的关键。迄今为止，RNA结合蛋白主要通过RNA纯化，然后通过质谱法鉴定。在这里，我们开发了incPRINT（细胞内蛋白质-RNA相互作用），这是一种高通量方法，用于识别通过可定量发光揭示的细胞内RNA-蛋白质相互作用。将incPRINT应用于长非编码RNA（lncRNA），我们鉴定了与lncRNA Firre和lncRNA Xist的三个功能不同区域特异性相互作用的RBP。incPRINT证实了先前已知的lncRNA-蛋白质相互作用，并鉴定了其他方法无法检测到的其他相互作用。重要的是，大多数incPRINT定义的相互作用是特定于大Xist转录本的单个功能区域。因此，我们提出了一种以RNA为中心的方法，该方法能够可靠地鉴定RNA区域特异性RBP，并且适用于任何感兴趣的RNA。
Regulatory RNAs exert their cellular functions through RNA-binding proteins (RBPs). Identifying RNA-protein interactions is therefore key for a molecular understanding of regulatory RNAs. To date, RNA-bound proteins have been identified primarily through RNA purification followed by mass spectrometry. Here, we develop incPRINT (in cell protein-RNA interaction), a high-throughput method to identify in-cell RNA-protein interactions revealed by quantifiable luminescence. Applying incPRINT to long noncoding RNAs (lncRNAs), we identify RBPs specifically interacting with the lncRNA Firre and three functionally distinct regions of the lncRNA Xist. incPRINT confirms previously known lncRNA-protein interactions and identifies additional interactions that had evaded detection with other approaches. Importantly, the majority of the incPRINT-defined interactions are specific to individual functional regions of the large Xist transcript. Thus, we present an RNA-centric method that enables reliable identification of RNA-region-specific RBPs and is applicable to any RNA of interest.