A 3-HYBRID SYSTEM TO DETECT RNA-PROTEIN INTERACTIONS IN-VIVO

RNA-protein interactions are pivotal in fundamental cellular processes such as translation, mRNA processing, early development, and infectio n 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-pr otein interactions in which the binding of a bifunctional RNA to each of two hybrid proteins activates transcription of a reporter gene in v ivo. We demonstrate that this three-hybrid system enables the rapid, p henotypic detection of specific RNA-protein interactions. As examples, we use the binding of the iron regulatory protein 1 (IRP1) to the iro n response element (IRE), and of HIV trans-activator protein (Tat) to the HIV trans-activation response element (TAR) RNA sequence. The thre e-hybrid assay we describe relies only on the physical properties of t he 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 thei r interactions.