Figure 1.  Visualization of Reporter mRNAs in Live Cells

(A) Schematic describing the constructs used in this approach. The system is comprised of two components, a reporter mRNA and a GFP-MS2 fusion protein. The GFP-MS2 was expressed under the control of the constitutive GPD promoter, while the reporter mRNA was under the control of the GAL promoter. The reporter mRNA contains six binding sites for the coat protein of the bacterial phage MS2. To avoid possible interference with translation and the function of the 3'UTR, the MS2-binding sites were introduced immediately after a translation termination codon. The 3'UTRs were either from the ASH1 gene, to induce mRNA localization at the bud tip, or from the ADHII gene, as control. In addition, a nuclear localization signal (NLS) followed by an HA tag was introduced at the N terminus of the fusion protein, so that that only the GFP protein that is bound to its target mRNA would be present in the cytoplasm.

(B) Live cells expressing the GFP-MS2 fusion protein and the acZ-MS2-ASH1 reporter mRNA. Arrows indicate some of the particles, usually in the bud. Bar, 5 µm.