Yeast Three Hybrid System
The three-hybrid system enables the detection of RNA-protein interactions in yeast using simple phenotypic assays. It was developed in collaboration with Stan Fields laboratory (University of Washington-Seattle).
Below, we provide three pertinent papers on the system, as well as include practical information including sequences and restriction maps of Hybrid RNA plasmids. We also include citations and links to recent published applications of the method.
If you have questions or improvements, please contact us.
Descriptions of the method
Original publication of the method [D. SenGupta, B. Zhang, B. Kraemer, P. Prochart, S. Fields and M. Wickens. 1996. A three-hybrid system for detecting RNA-protein interactions. Proc. Natl. Acad. Sci. 93, 8496-8501] [Pub Med]
David Bernstein, Natascha Buter, Craig Stumpf and Marvin Wickens. 2002. Analyzing mRNA-protein complexes using a yeast three-hybrid system: methods and applications. Methods 2, 123-41 [Pub Med]
Stumpf C, Opperman L, Wickens M. (2008) Analysis of RNA–Protein Interactions Using a Yeast Three-Hybrid System. Methods in Enzymology, Vol 449.
Practical information [unpublished].
Here are the sequences of the plasmids used in the three-hybrid system. If you have made improvement on the system, or have suggestions on how to improve the system, please contact us.
plllA/MS2-1 and its map (postscript version for printout)
plllA/MS2-2 and its map (postscript version for printout)
Selected applications and improvement
Hook, B., D. Bernstein, B. Zhang, and M. Wickens. (2005). RNA-protein interactions in the yeast three-hybrid system: affinity, sensitivity and enhanced library screening. RNA, 11, 227-233.[Pub Med]
Z. F. Wang, M. L. Whitfield, T. C. I. Ingledue, Z. Dominski, and W. F. Marzluff. 1996. The protein that binds the 3' end of histone mRNA: a novel RNA-binding protein required for histone pre-mRNA processing, Genes Dev 10, 3028 (1996) [PubMed].
F. Martin, A. Schaller, S. Eglite, D. Schumperli, and B. Muller. 1997. The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein, EMBO J 16, 769 [PubMed].
B. Zhang, M. Gallegos, A. Puoti, E. Durkin, S. Fields, J. Kimble, and M. P. Wickens. 1997. A conserved RNA-binding protein that regulates sexual fates in the C. elegans hermaphrodite germ line, Nature 390, 477 (1997) [PubMed].
E. Bacharach, and S. P. Goff. 1998. Binding of the human immunodeficiency virus type 1 Gag protein to the viral RNA encapsidation signal in the yeast three-hybrid system, Journal of Virology 72, 6944-6969 [PubMed].
A. Dahanukar, J. A. Walker, and R. P. Wharton. 1999. Smaug, a novel RNA-binding protein that operaties a translational switch in Drosophila, Molecular Cell 4, 1-20 [PubMed].
S. W. Fewell, and J. L. Woolford. 1999. Ribosomal protein S14 of Saccharomyces cerivisiae regulates its expression by binding to RPS14B premRNA and to 18S rRNA, Mol. Cell. Biol. 19, 826-834 [PubMed].
E. Jan, C. K. Motzny, L. E. Graves, and E. B. Goodwin. 1999. The star protein GLD-1 is a translational regulator of sexual identity in c. elegans., EMBO J. 18, 259-269 [PubMed].
E. G. Lee, A. Yeo, B. Kraemer, M. Wickens, and M. L. Linial. 1999. The Gag domains required for avian retroviral RNA encapsidation determined by using two independent assays, Journal of Virology 73, 6282-6292 [PubMed].
Y. W. Park, S. L. Tan, and M. G. Katze. 1999. Differential sensitivity to 5-fluoro-orotic acid as a screen for bait RNA-independent false positives in a yeast three-hybrid system, Biotechniques 26, 1102-1106 [PubMed].
Y.W. Park, J. Wilusz, and M. G. Katze. 1999. Regulation of eukaryotic protein synthesis: selective influenze viral mRNA translation is mediated by cellular RNA binding protein GRSF-1, Proc.Natl Acad. Sci. 96, 6694-6699 [PubMed].
D. SenGupta, M. Wickens, and S. Fields. 1999. Identification of RNAs that bind to a specific protein using the yeast three-hybrid system. RNA 5, 596-601 [PubMed].
J. Sonoda and R.P. Wharton. 1999. Recruitment of Nanos to hunchback mRNA by Pumilio. Genes Dev. 13: 2704-2712 [PubMed].
Z. F. Wang, T. C. Ingldue, R. Sanchez, and W. F. Marzluff. 1999. Two Xenopus proteins that bind the 3' end of histone mRNA: implications for translational control of protein synthesis during oogenesis, Mol. Cell. Biol. 19, 835-845 [PubMed].