The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells.
The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells.
复制标题
RNA-KISS 的开发，一种用于检测活哺乳动物细胞中 RNA-蛋白质相互作用的哺乳动物三杂交方法。
DOI：
10.1021/acs.jproteome.0c00068
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发表时间：
2020
影响因子：
4.4
通讯作者：
Tavernier,Jan
中科院分区：
文献类型：
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作者：
Lemmens,Irma;Jansen,Sander;deRouck,Steffi;deSmet,Anne-Sophie;Defever,Dieter;Neyts,Johan;Dallmeier,Kai;Tavernier,Jan
RNA-蛋白质相互作用对于mRNA和非编码RNA功能的调节是必不可少的，并且涉及许多疾病，例如癌症和神经退行性疾病。一种可以在蛋白质组范围内检测活哺乳动物细胞中RNA-蛋白质相互作用的方法将是识别和研究这些相互作用的重要资产。在这里，我们表明哺乳动物双杂交蛋白质-蛋白质检测方法KISS（激酶底物传感器）和酵母RNA三杂交方法的组合，利用MS 2 RNA和MS 2外壳蛋白之间的特异性相互作用，能够检测RNA-蛋白质相互作用活哺乳动物细胞中的相互作用。为了概念证明，我们使用登革热病毒（DENV）的亚基因组黄病毒RNA（sfRNA）作为诱饵，所述亚基因组黄病毒RNA（sfRNA）是一种源自DENV基因组的高度结构化的非编码RNA，已知其靶向参与先天免疫和抗病毒防御的宿主细胞蛋白。使用RNA-KISS，我们可以证实先前建立的DDX 6的RNA结合结构域与DENV sfRNA之间的相互作用。最后，我们对DENV sfRNA结合宿主因子进行了人类蛋白质组范围的筛选，鉴定了几种已知的黄病毒宿主因子，如DDX 6和PACT，进一步验证了RNA-KISS方法作为一种稳健的高通量基于细胞的RNA-蛋白质相互作用筛选工具。
RNA–protein interactions are essential for the regulation of mRNA and noncoding RNA functions and are implicated in many diseases, such as cancer and neurodegenerative disorders. A method that can detect RNA–protein interactions in living mammalian cells on a proteome-wide scale will be an important asset to identify and study these interactions. Here we show that a combination of the mammalian two-hybrid protein–protein detection method KISS (kinase substrate sensor) and the yeast RNA three-hybrid method, utilizing the specific interaction between the MS2 RNA and MS2 coat protein, is capable of detecting RNA–protein interactions in living mammalian cells. For conceptional proof we used the subgenomic flavivirus RNA (sfRNA) of the dengue virus (DENV), a highly structured noncoding RNA derived from the DENV genome known to target host cell proteins involved in innate immunity and antiviral defense, as bait. Using RNA-KISS, we could confirm the previously established interaction between the RNA-binding domain of DDX6 and the DENV sfRNA. Finally, we performed a human proteome-wide screen for DENV sfRNA-binding host factors, identifying several known flavivirus host factors such as DDX6 and PACT, further validating the RNA-KISS method as a robust and high-throughput cell-based RNA–protein interaction screening tool.