SenGupta D J, Zhang B, Kraemer B, Pochart P, Fields S, Wickens M
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook 11794, USA.
Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8496-501. doi: 10.1073/pnas.93.16.8496.
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.
RNA与蛋白质的相互作用在诸如翻译、mRNA加工、早期发育以及RNA病毒感染等基本细胞过程中起着关键作用。然而，尽管这些相互作用至关重要，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与蛋白质的相互作用，其中双功能RNA与两种杂交蛋白各自的结合在体内激活报告基因的转录。我们证明这种三杂交系统能够对特定RNA与蛋白质的相互作用进行快速的表型检测。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反式激活应答元件（TAR）RNA序列的结合。我们所描述的三杂交测定仅依赖于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可能具有广泛的应用。
