A three-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 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.
SenGupta
D J
DJ
Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook 11794, USA.
Zhang
B
B
Kraemer
B
B
Pochart
P
P
Fields
S
S
Wickens
M
M
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D015696:Gene Products, tat / Q000378:metabolism; D005822:Genetic Vectors; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012641:Selection, Genetic
D017931:DNA Primers; D015696:Gene Products, tat; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D035941:Iron Regulatory Protein 1