LOCALIZATION OF ASH1 MESSENGER-RNA PARTICLES IN LIVING YEAST

ASH1 mRNA localizes to the bud tip in Saccharomyces cerevisiae to esta blish 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 (un translated region) sequences. The SHE mutants disrupt RNA and particle localization and SHE 2 and 3 mutants inhibit particle formation as we ll. Both She3myc and She1myc colocalized with the particle. Video micr oscopy demonstrated that She1p/Myo4p moved particles to the bud tip at 200-440 nm/sec. Therefore, the ASH1 3'UTR-dependent particle serves a s a marker for RNA transport and localization.