A three-hybrid system to detect RNA-protein interactions in vivo
A three-hybrid system to detect RNA-protein interactions in vivo
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DOI：
10.1073/pnas.93.16.8496
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发表时间：
1996-08-06
影响因子：
11.1
通讯作者：
Wickens, M
中科院分区：
文献类型：
--
作者：
SenGupta, DJ;Zhang, BL;Wickens, M
RNA - 蛋白质相互作用在基本的细胞过程中至关重要，例如翻译、mRNA加工、早期发育以及RNA病毒感染。然而，尽管这些相互作用非常重要，但在体内快速分析它们的方法却很少。我们描述了一种酵母遗传学方法来检测和分析RNA - 蛋白质相互作用，其中双功能RNA与两种杂交蛋白中的每一种的结合会激活体内报告基因的转录。我们证明这种三杂交系统能够快速、表型地检测特定的RNA - 蛋白质相互作用。例如，我们利用铁调节蛋白1（IRP1）与铁反应元件（IRE）的结合，以及HIV反式激活蛋白（Tat）与HIV反式激活反应元件（TAR）RNA序列的结合。我们所描述的三杂交分析仅依赖于RNA和蛋白质的物理性质，而不依赖于它们的天然生物学活性；因此，它可能在RNA结合蛋白和RNA的鉴定以及它们相互作用的详细分析中具有广泛的应用。
RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infection by RNA viruses. However, in spite of the central importance of these interactions, few approaches are available to analyze them rapidly in vivo. We describe a yeast genetic method to detect and analyze RNA-protein interactions in which the binding of a bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in vivo. We demonstrate that this three-hybrid system enables the rapid, phenotypic detection of specific RNA-protein interactions. As examples, we use the binding of the iron regulatory protein 1 (IRP1) to the iron response element (IRE), and of HIV trans-activator protein (Tat) to the HIV trans-activation response element (TAR) RNA sequence. The three-hybrid assay we describe relies only on the physical properties of the RNA and protein, and not on their natural biological activities; as a result, it may have broad application in the identification of RNA-binding proteins and RNAs, as well as in the detailed analysis of their interactions.