Beach D L, Salmon E D, Bloom K
Department of Biology, University of North Carolina, 212 Coker Hall CB3280, Chapel Hill, North Carolina, 27599-3280, USA.
Curr Biol. 1999 Jun 3;9(11):569-78. doi: 10.1016/s0960-9822(99)80260-7.
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.
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 'gRNAASH1'). The gRNAASH1 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 Bnilp/She5p, which are involved in polarity establishment and actin organization, gRNAASH1 migrated to the bud but failed to remain at the bud tip. These studies reveal discrete transport and anchoring steps in mRNA localization.
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.
真核细胞将特定的信使核糖核酸（mRNA）定位到细胞的特定区域，以此作为隔离蛋白质的一种方式。3'非翻译区（3'UTR）内的信号通过肌动蛋白和微管细胞骨架系统促进mRNA的定位。最近，酿酒酵母中的一种mRNA，即ASH1，被证明可聚集成一个离散的颗粒，并维持在芽尖处。五个基因SHE1 - SHE5中的突变会导致ASH1转录本的颗粒形成和/或定位缺陷。mRNA运输目的地的因子仍有待确定。
我们开发了一种系统，用于在活酵母中用绿色荧光蛋白（GFP）标记mRNA，并跟踪mRNA运动和定位的动态过程。组成型表达一种包含与ASH1 3'UTR相邻的噬菌体MS2外壳蛋白结合位点的ASH1 mRNA，使我们能够用MS2外壳蛋白 - GFP融合蛋白（统称为“gRNAASH1”）观察ASH1 mRNA。gRNAASH1在小芽到中芽及大芽细胞中局限于芽尖，在细胞分裂前迁移到芽颈，然后迅速重新定位到芽生长的起始位点。在交配过程中，它也定位于极性生长区域。在缺乏参与极性建立和肌动蛋白组织的Bud6p/Aip3p或Bnilp/She5p的细胞中，gRNAASH1迁移到芽中，但未能停留在芽尖。这些研究揭示了mRNA定位中离散的运输和锚定步骤。
在整个营养和交配细胞周期中，ASH1 mRNA都维持在极性生长位点。需要Bud6p/Aip3p和Bni1p/She5p将转录本维持在皮质芽帽处。
