RNA TRANSPORT AND LOCALIZATION IN YEAST IN SITU

超出所提供的空间。许多mRNA种类定位于细胞内。前缘的-肌动蛋白合成促进了细胞的极性和运动性。在酵母中，ASH1 mRNA定位于芽尖，是母芽之间不对称基因表达所必需的，允许交配类型转换。一个主要的问题是mRNA定位究竟是如何发生的，以及涉及哪些因素。为了解决这个问题，开发了一种可视化活细胞内mrna运动的方法。使用融合RNA结合蛋白MS2的GFP报告系统并将MS2序列引入报告RNA中，可以在活酵母细胞中观察到ASH1 mRNA的运动（Bertrand等人，Mol. Cell, 1998）。这种方法明确地表明，mRNA形成了一个在肌动蛋白电缆上移动的颗粒和两种蛋白质；She2p是一种结合ASH1 mRNA中四个zip编码中的每一个的蛋白，而肌球蛋白Shelp则由第三种蛋白She3p连接（Long, et al., EMBO J. 19: 6592,2000）。我们建议分离“locasome”，即由ASH1邮编形成的粒子。由于绿色荧光蛋白标记，该粒子将在光学显微镜下被视觉识别。通过分离该结构，我们将能够识别与其（假设的）核形成、输出、与肌凝蛋白马达的关联、芽尖锚定和翻译调节相关的各种蛋白质。在半乳糖诱导后，这些基因将在GFP颗粒定位试验中通过突变或缺失进行检测。利用高速显微镜对GFP颗粒进行可视化观察，可以捕捉到转录释放和向核孔、输出和偶联到细胞质丝的快速运动过程。增加RNA上MS2结合位点的数量将使我们能够检测单个RNA分子。使用这些方法，我们将有机会以高空间和时间分辨率确定蛋白质表达的定位和调节成分在途径中的位置。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. Numerous mRNA species localize within cells. The synthesis ofbeta-actin at the leading edge facilitates cell polarity and motility. In yeast, ASH1 mRNA localizes to the bud tip and is necessary for asymmetric gene expression between the mother and the bud, allowing mating type switching. A major question has been exactly how mRNA localization occurs and what factors are involved. In order to address this, a method for visualizing the movement ofmRNAs within living cells was developed. ASH1 mRNA movements are observed in living yeast cells using a GFP reporter system fused to the RNA-binding protein MS2 and introduction of MS2 sequences into the reporter RNA (Bertrand et al, Mol. Cell 2:437, 1998). This approach showed unequivocally that the mRNA formed a particle that moved on actin cables and that two proteins; She2p, a protein that binds each of the four zipcodes in the ASH1 mRNA, and the myosin, Shelp, are bridged by a third protein, She3p (Long, et al., EMBO J. 19:6592, 2000). We propose to isolate the "locasome", the particle formed by the ASH1 zipcodes. Because of the GFP tagging, this particle will be visually identifiable by light microscopy. By isolating this structure, we will be able to identify the various proteins associated with its (hypothesized) nuclear formation, export, association with the myosin motor, anchoring at the bud tip and translational regulation. These genes will be tested by mutation or deletion in the GFP particle localization assay, after induction by galactose. The visualization of the GFP particle using high-speed microscopy will capture the rapid movements during transcriptional release and movement to the nuclear pores, export and coupling to the cytoplasmic filaments. Increasing the number of MS2 binding sites on the RNA will allow us to detect single RNA molecules. Using these approaches, we will have the opportunity to identify where in the pathway the components of localization and regulation of protein expression operate, with high spatial and temporal resolution. PERFORMANCE SITE ========================================Section End===========================================