Localization of ASH1 mRNA particles in living yeast

Abstract

ASH1 mRNA localizes to the bud tip in Saccharomyces cerevisiae to establish asymmetry of HO expression, important for mating type switching. To visualize real time localization of the mRNA in living yeast cells, green fluorescent protein (GFP) was fused to the RNA-binding protein MS2 to follow a reporter mRNA containing MS2-binding sites. Formation and localization of a GFP particle in the bud required ASH1 3′UTR (untranslated region) sequences. The SHE mutants disrupt RNA and particle localization and SHE 2 and 3 mutants inhibit particle formation as well. Both She3myc and Shelmyc colocalized with the particle. Video microscopy demonstrated that She1p/Myo4p moved particles to the bud tip at 200-440 nm/sec. Therefore, the ASH1 3′UTR-dependent particle serves as a marker for RNA transport and localization.