The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells.,Journal of Proteome Research

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The Development of RNA-KISS, a Mammalian Three-Hybrid Method to Detect RNA-Protein Interactions in Living Mammalian Cells.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2020-03-27 , DOI: 10.1021/acs.jproteome.0c00068
Irma Lemmens _{1,

2} , Sander Jansen ₃ , Steffi de Rouck _{1,

2} , Anne-Sophie de Smet _{1,

2} , Dieter Defever _{1,

2} , Johan Neyts ₃ , Kai Dallmeier ₃ , Jan Tavernier _{1,

2,

4}

Affiliation

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.

中文翻译：

RNA-KISS的发展，一种检测哺乳动物细胞中RNA-蛋白质相互作用的哺乳动物三杂交方法。

RNA与蛋白质的相互作用对于调节mRNA和非编码RNA的功能至关重要，并且与许多疾病有关，例如癌症和神经退行性疾病。一种可以检测整个蛋白质组规模的哺乳动物活细胞中RNA-蛋白质相互作用的方法将是鉴定和研究这些相互作用的重要资产。在这里，我们展示了哺乳动物两杂交蛋白-蛋白质检测方法KISS（激酶底物传感器）和酵母RNA三杂交方法的结合，利用MS2 RNA和MS2外壳蛋白之间的特异性相互作用，能够检测RNA –哺乳动物活细胞中的蛋白质相互作用。为了进行概念验证，我们使用了登革热病毒（DENV）的亚基因组黄病毒RNA（sfRNA），从DENV基因组衍生的高度结构化的非编码RNA，已知它靶向与先天免疫和抗病毒防御相关的宿主细胞蛋白，如诱饵。使用RNA-KISS，我们可以确认DDX6的RNA结合结构域与DENV sfRNA之间先前建立的相互作用。最后，我们对DENV sfRNA结合宿主因子进行了全蛋白质组筛选，鉴定了几种已知的黄病毒宿主因子，例如DDX6和PACT，进一步验证了RNA-KISS方法是一种可靠且高通量的基于细胞的RNA蛋白互动筛选工具。

更新日期：2020-03-27

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