Abstract
Localized mRNA translation is involved in cell-fate determination, polarization, and morphogenesis in eukaryotes. While various tools are available to examine mRNA localization, no easy and quick method has allowed for the visualization of endogenously expressed mRNAs in vivo. We describe a simple method (m-TAG) for PCR-based chromosomal gene tagging that uses homologous recombination to insert binding sites for the RNA-binding MS2 coat protein (MS2-CP) between the coding region and 3′-untranslated region of any yeast gene. Upon co-expression of MS2-CP fused with GFP, specific endogenously expressed mRNAs can be visualized in vivo for the first time. This method allows for the easy examination of mRNA localization using fluorescence microscopy and leaves the yeast cells amenable for further genetic analysis.
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Bashirullah, A., Cooperstock, R. L., and Lipshitz, H. D. (1998) RNA localization in development Annu. Rev. Biochem. 67, 335–394.
Gonsalvez, G. B., Urbinati, C. R., and Long, R. M. (2005) RNA localization in yeast: moving towards a mechanism Biol. Cell 97, 75–86.
Kloc, M., Zearfoss, N. R., and Etkin, L. D. (2002) Mechanisms of subcellular mRNA localization Cell 108, 533–544.
Haim, L., Zipor, G., Aronov, S., and Gerst, J. E. (2007) A genomic integration method to visualize localization of endogenous mRNAs in living yeast Nat. Methods 4, 409–412.
Haim-Vilmovsky, L., and Gerst, J. E. (2009) m-TAG: a PCR-based genomic integration method to visualize the localization of specific endogenous mRNAs in vivo in yeast Nat. Protoc. 4, 1274–1284.
Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., et al. (2002) Functional profiling of the Saccharomyces cerevisiae genome Nature 418, 387–391.
Ghaemmaghami, S., Huh, W. K., Bower, K., Howson, R. W., Belle, A., et al. (2003) Global analysis of protein expression in yeast Nature 425, 737–741.
Huh, W. K., Falvo, J. V., Gerke, L. C., Carroll, A. S., Howson, R. W., et al. (2003) Global analysis of protein localization in budding yeast Nature 42 5, 686–691.
Rose, M. D., Winston, F., Hieter, P. (1990) Methods in Yeast Genetics, A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Acknowledgements
This work was supported by grants to J.E.G. from the Minerva Foundation, Germany, and the Y. Leon Benoziyo Center for Molecular Medicine, Center for Scientific Excellence, and Kahn Center for Systems Biology, Weizmann Institute of Science. J.E.G. holds the Besen-Brender Chair in Microbiology and Parasitology.
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Haim-Vilmovsky, L., Gerst, J.E. (2011). Visualizing Endogenous mRNAs in Living Yeast Using m-TAG, a PCR-Based RNA Aptamer Integration Method, and Fluorescence Microscopy. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_15
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DOI: https://doi.org/10.1007/978-1-61779-005-8_15
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