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A genomic integration method to visualize localization of endogenous mRNAs in living yeast

Nature Methods volume 4, pages 409–412 (2007)Cite this article

Abstract

mRNA localization may be an important determinant for protein localization. We describe a simple PCR-based genomic-tagging strategy (m-TAG) 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 (UTR) of any yeast gene. Upon coexpression of MS2-CP fused with GFP, we demonstrate the localization of endogenous mRNAs (ASH1, SRO7, PEX3 and OXA1) in living yeast (Saccharomyces cerevisiae).

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Figure 1: A schematic representation of the MS2 loop genomic-tagging strategy (m-TAG).
Figure 2: Visualization of endogenous ASH1 mRNA localization in vivo.
Figure 3: Visualization of other endogenous yeast mRNAs in vivo.

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Acknowledgements

We thank K. Bloom, P. Brennwald, J. Brickner, M. Longtine, S. Michaelis, R. Singer and D. Stillman for the generous gifts of reagents; A. Cohen and I. Goldshtein for technical assistance. This work was generously supported by a grant from the Kahn Fund for Systems Biology, Weizmann Institute of Science. 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, 76100, Israel

    Liora Haim, Gadi Zipor, Stella Aronov & Jeffrey E Gerst

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  1. Liora Haim
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  2. Gadi Zipor
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  3. Stella Aronov
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  4. Jeffrey E Gerst
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Contributions

Experimental design was by L.H., S.A. and J.E.G.; reagent preparation by L.H. and G.Z.; data collection, analysis, figure preparation and text by L.H. and J.E.G.

Corresponding author

Correspondence to Jeffrey E Gerst.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

PCR analysis and detection of MS2 loop integration and marker excision. (PDF 83 kb)

Supplementary Fig. 2

Sro7 is functional in SRO7::loxP::MS2L::SRO73′-UTR cells. (PDF 157 kb)

Supplementary Table 1

Oligonucleotides used in this study. (PDF 12 kb)

Supplementary Table 2

Yeast strains used in this study. (PDF 46 kb)

Supplementary Methods (PDF 116 kb)

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Haim, L., Zipor, G., Aronov, S. et al. A genomic integration method to visualize localization of endogenous mRNAs in living yeast. Nat Methods 4, 409–412 (2007). https://doi.org/10.1038/nmeth1040

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