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m-TAG: a PCR-based genomic integration method to visualize the localization of specific endogenous mRNAs in vivo in yeast

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Abstract

This protocol describes m-TAG, a novel method for the visualization of endogenously expressed mRNAs in live yeast (Saccharomyces cerevisiae). First, a gene of interest is tagged with multiple binding sites for the RNA-binding MS2 coat protein (MS2-CP), using a PCR-based genomic-tagging strategy and homologous recombination. Next, MS2-CP fused to GFP(x3) is expressed in cells; binding of this fusion protein to the tagged mRNA yields an RNA granule that can be visualized by fluorescence microscopy. While existing methods necessitate cell fixation (for in situ hybridization) or the detection of exogenously expressed mRNAs (from plasmids), or give transient signals (i.e., with fluorescent hybridization probes), m-TAG allows for the robust and stable visualization of endogenously expressed mRNAs in vivo and facilitates the study of mRNA dynamics under different growth conditions. The m-TAG procedure is simple, easy to perform and takes <3 weeks to yield cultured yeast strains for mRNA visualization.

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Figure 1: m-TAG, a method for visualizing endogenous mRNAs in living yeast cells.
Figure 2: The m-TAG gene-tagging strategy: primers and protocol.
Figure 3: Visualization of endogenous mRNAs in vivo.

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Acknowledgements

This work was supported by grants from the Kahn Center for Systems Biology and Y. Leon Benoziyo Institute for Molecular Medicine, Weizmann Institute of Science, Israel, and the Minerva Foundation, Germany. J.E.G. holds the Henry Kaplan Chair in Cancer Research.

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Authors and Affiliations

  1. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Liora Haim-Vilmovsky & Jeffrey E Gerst

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  1. Liora Haim-Vilmovsky
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  2. Jeffrey E Gerst
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Text and figure preparation was done by L.H.-V. and J.E.G.

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Correspondence to Jeffrey E Gerst.

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Haim-Vilmovsky, L., Gerst, J. 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 (2009). https://doi.org/10.1038/nprot.2009.115

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