Identification of RNAs that bind to a specific protein using the yeast three-hybrid system.
We have adapted the yeast three-hybrid system to identify RNA ligands for an RNA-binding protein. In this assay system, a protein-RNA interaction is detected by the reconstitution of a transcriptional activator using two hybrid proteins and a hybrid RNA. The RNA molecule is tethered to the promoter of a reporter gene by binding to a hybrid protein consisting of the bacteriophage MS2 coat protein fused to the DNA-binding protein LexA; the RNA-binding domain to be analyzed is fused to the transcriptional activation domain of the yeast Gal4 protein; and the bifunctional RNA consists of binding sites for the coat protein and for the other RNA-binding domain. We built an RNA library such that short fragments of genomic DNA from yeast were transcribed in yeast together with binding sites for the coat protein. We screened this hybrid RNA library for RNAs that bound to the yeast Snp1 protein, a homolog of the human U1-70K protein. The screen yielded as the strongest positive the fragment of U1 RNA that contains loop I, which is known to bind to Snp1 in U1 snRNP. We also identified four other RNA ligands that produced weaker three-hybrid signals, suggesting lower affinities for Snp1 as compared to U1 RNA. In addition, this search also yielded a set of RNA sequences that can activate transcription on their own when bound to a promoter through a protein interaction.
Sengupta
D J
DJ
Department of Genetics, University of Washington, Seattle 98195, USA.
Wickens
M
M
Fields
S
S
D002213:Capsid / Q000235:genetics; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics*; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D008024:Ligands; D011401:Promoter Regions, Genetic / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008024:Ligands; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D014157:Transcription Factors