Abstract
Localization of messenger RNA (mRNA) is a process used by eukaryotes to control the spatio-temporal expression of proteins involved in cellular motility, asymmetric cell division, or polarized cell growth. A better understanding of this process relies on methods to detect specifically the position of an mRNA in fixed or living cells. This chapter presents methods to visualize mRNA in both fixed and living yeast Saccharomyces cerevisiae. In fixed cells, position of mRNAs can be assessed by using Fluorescent In Situ Hybridization (FISH) that consists of the hybridization of fluorescent probes that target a specific transcript in situ. In living cells, dynamics of mRNAs can be monitored using a bipartite system composed of MS2 stem-loops inserted in the mRNA of interest. These stem-loops are recognized specifically by the MS2 RNA-binding protein, fused to a fluorescent protein. In vivo association between the reporter (fluorescent MS2 protein) and the MS2-tagged mRNA reconstitutes active fluorescent ribonucleoparticles that can be followed by live cell imaging. Detailed protocols for the realization of these methods are provided and several technical considerations are discussed. Together, these methods provide very robust tools to determine the intracellular position and dynamics of your mRNA of interest in yeast.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Amberg, D. C., Goldstein, A.L., Cole, C.N. (1992) Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA. Genes Dev. 6, 1173–89.
Long, R. M., Singer, R. H., Meng, X., Gonzalez, I., Nasmyth, K., and Jansen, R.-P. (1997) Mating Type Switching in Yeast Controlled by Asymmetric Localization of ASH1 mRNA. Science 277, 383–87.
Zenklusen, D., Larson, D. R., and Singer, R. H. (2008) Single-RNA counting reveals alternative modes of gene expression in yeast. Nat Struct Mol Biol 15, 1263–71.
Long, R. M., Elliott, D.J., Stutz, F., Rosbash, M., Singer, R.H. (1995) Spatial consequences of defective processing of specific yeast mRNAs revealed by fluorescent in situ hybridization. RNA 1, 1071–78.
Saavedra, C., Tung, K. S., Amberg, D. C., Hopper, A. K., and Cole, C. N. (1996) Regulation of mRNA export in response to stress in Saccharomyces cerevisiae. Genes & Development 10, 1608–20.
Garcia, M., Darzacq, X., Delaveau, T., Jourdren, L., Singer, R. H., and Jacq, C. (2007) Mitochondria-associated Yeast mRNAs and the Biogenesis of Molecular Complexes. Mol. Biol. Cell 18, 362–68.
Aronov, S., Gelin-Licht, R., Zipor, G., Haim, L., Safran, E., and Gerst, J. E. (2007) mRNAs Encoding Polarity and Exocytosis Factors Are Cotransported with the Cortical Endoplasmic Reticulum to the Incipient Bud in Saccharomyces cerevisiae. Mol. Cell. Biol. 27, 3441–55.
Chartrand, P., Singer, R.H., and Long, R.M. (2000) Sensitive and high-resolution detection of RNA in situ. Methods in Enzymol 318, 493–506.
Bertrand, E., Chartrand, P., Schaefer, M., Shenoy, S.M., Singer, R.H., and Long, R.M (1998) Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–45.
Beach, D. L., Salmon, E. D., and Bloom, K. (1999) Localization and anchoring of mRNA in budding yeast. Current Biology 9, 569–78.
Brodsky, A. S., and Silver, P. A. (2000) Pre-mRNA processing factors are required for nuclear export. RNA 6, 1737-49.
Daigle, N., and Ellenberg, J. (2007) λN-GFP: an RNA reporter system for live-cell imaging. Nat Meth 4, 633–36.
Bernardi, A., Spahr, P.F. (1972) Nucleotide sequence at the binding site for coat protein on RNA of bacteriophage R17. Proc Natl Acad Sci USA. 69, 3033–37.
Lim, F., and Peabody, D. S. (1994) Mutations that increase the affinity of a translational repressor for RNA. Nucl. Acids Res. 22, 3748–52.
Talbot, S. J., Goodman, S., Bates, S. R. E., Fishwick, C. W. G., and Stockley, P. G. (1990) Use of synthetic oligoribonucleotides to probe RNA-protein interactions in the MS2 translational operator complex. Nucl. Acids Res. 18, 3521–28.
Rose, M. D., Winston, F., and Hieter, P. (1990) Methods in yeast genetics. A laboratory course manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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 Meth 4, 409–12.
Schmid, M., Jaedicke, A., Du, T.-G., and Jansen, R.-P. (2006) Coordination of Endoplasmic Reticulum and mRNA Localization to the Yeast Bud. Current Biology 16, 1538–43.
Gallardo, F., Olivier, C., Dandjinou, A.T., Wellinger, R.J., Chartrand, P. (2008) TLC1 RNA nucleo-cytoplasmic trafficking links telomerase biogenesis to its recruitment to telomeres. The Embo Journal 27, 748–57.
Acknowledgments
The authors thank Dr. Emmanuelle Querido for critical reading of the manuscript and Zhifa Shen for sharing results. This work is supported by a grant from the Natural Sciences and Engineering Research Council of Canada. FG is supported by a fellowship from the Terry Fox Foundation from the National Cancer Institute of Canada. PC is a Senior Scholar from the Fond de Recherche en Santé du Québec (FRSQ).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Gallardo, F., Chartrand, P. (2011). Visualizing mRNAs in Fixed and Living Yeast Cells. 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_13
Download citation
DOI: https://doi.org/10.1007/978-1-61779-005-8_13
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-004-1
Online ISBN: 978-1-61779-005-8
eBook Packages: Springer Protocols