Identification and detection of RNA-RNA interactions using the yeast RNA hybrid system.
To test RNA-RNA interactions in cells, we developed a yeast RNA hybrid system derived from the yeast three-hybrid system. In this setup, the activation of a reporter gene (HIS3 or lacZ) is dependent on the interaction of two RNAs. One ('RNA X') is fused to MS2 RNA, forming the bait, which binds to a fusion protein composed of the MS2 coat and the LexA proteins. The second ('RNA Y') is fused to an RNA-based transcriptional activator (m26-11), forming the prey. If prey (RNA Y) binds to bait (RNA X), the m26-11 transcriptional activator is tethered to the promoter of the reporter genes. This protocol describes how to use this RNA hybrid system. In addition to testing RNA-RNA interactions, it can also be used to screen RNA libraries to identify new interaction partners or for mutational analysis of two known interaction partners.
Piganeau
Nicolas
N
Institute of Biochemistry, University of Hamburg, Martin Luther King Platz 6, 20146 Hamburg, Germany.
Schroeder
Renée
R
D020029:Base Pairing; D001483:Base Sequence; D009154:Mutation; D012313:RNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D020798:Two-Hybrid System Techniques
D012313:RNA