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The Yeast Three-Hybrid System for Screening RNA-Binding Proteins in Plants

  • Sung Ki Cho
  • David J. HannapelEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1794)

Abstract

Yeast-hybrid methods have been successfully applied for screening interacting partners of DNAs or proteins. A yeast-based method, the yeast three-hybrid system, using a chimeric protein of a DNA-binding domain (LexA or GAL4BD) with a protein (MS2 coat protein or HIV Rev. M10) having a hybrid RNA at the 3′ end of a target RNA sequence, has been developed for screening RNA-binding proteins. When the target RNA interacts with RNA-binding proteins fused with an activation domain (AD), yeast cells having all the interacting components can survive on selection media, and interacting reporters, HIS3 and LacZ, are activated. Based on this selection, interaction can be easily monitored and detected by simple biochemical assays. The in vivo screening strategy has been widely applied for characterizing and evaluating specific interactions between target RNAs and RNA-binding proteins. Here, we describe a library screening strategy for isolating RNA-binding proteins of select target RNAs using the yeast three-hybrid method. We also describe strategies to verify binding specificity using both a yeast-dependent reporter system and a yeast-independent method, in vivo RNA immunoprecipitation (RIP).

Key words

Yeast hybridization RNA-binding protein RNA–protein interaction RNA electrophoretic mobility shift assay RNA immunoprecipitation 

Notes

Acknowledgment

Thanks to Drs. Il-Ho Kang and Tyrell Carr for their significant contributions to this methods report. Research for this work was supported by an award from the National Science Foundation Plant Genome Research Program to DH (Grant No. 0820659).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.The Department of Biochemistry, Cellular, and Molecular BiologyUniversity of TennesseeKnoxvilleUSA
  2. 2.Plant Biology MajorIowa State UniversityAmesUSA

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