Visualizing mRNAs in fixed and living yeast cells.

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.