Localization and anchoring of mRNA in budding yeast

Background: Eukaryotic cells localize selected mRNAs to a region of the cell as a means to sequester proteins. Signals within the 3' untranslated region (3'UTR) facilitate mRNA localization by both actin and microtubule cytoskeletal systems. Recently, an mRNA in the yeast Saccharomyces cerevisiae, ASH1, was shown to coalesce into a discrete particle that is maintained at the bud tip. Mutations in five genes, SHE1 -SHE5, cause defects in particle formation and/or localization of the ASH1 transcript. Factors at the destination of the mRNA transport remain to be identified. Results: We have developed a system to label mRNA in living yeast with green fluorescent protein (GFP) and follow the dynamics of mRNA movement and localization. Constitutively expressing an ASH1 mRNA containing the bacteriophage MS2 coat-protein binding site adjacent to the ASH1 3'UTR allowed us to visualize ASH1 mRNA with an MS2-coat-protein-GFP fusion protein (together denoted gRNA(ASH1)). The gRNA(ASH1) was restricted to the bud tip in small to large budded cells, migrated to the bud neck prior to cell separation and then rapidly relocalized to the incipient site of bud growth. It also localized to regions of polarized growth during mating. In cells lacking Bud6p/Aip3p or Bni1p/She5p, which are involved in polarity establishment and actin organization, gRNA(ASH1) migrated to the bud but failed to remain at the bud tip. These studies reveal discrete transport and anchoring steps in mRNA localization, Conclusions: The ASH1 mRNA was maintained at sites of polarized growth throughout the vegetative and mating cell cycles, Bud6p/Aip3p and Bni1p/She5p are required to maintain the transcript at the cortical bud cap.