MUTATION OR DELETION OF THE SACCHAROMYCES-CEREVISIAE RAT3/NUP133 GENE CAUSES TEMPERATURE-DEPENDENT NUCLEAR ACCUMULATION OF POLY(A)(+) RNA AND CONSTITUTIVE CLUSTERING OF NUCLEAR-PORE COMPLEXES
MUTATION OR DELETION OF THE SACCHAROMYCES-CEREVISIAE RAT3/NUP133 GENE CAUSES TEMPERATURE-DEPENDENT NUCLEAR ACCUMULATION OF POLY(A)(+) RNA AND CONSTITUTIVE CLUSTERING OF NUCLEAR-PORE COMPLEXES
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DOI：
10.1091/mbc.6.4.401
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发表时间：
1995-04-01
影响因子：
3.3
通讯作者：
COLE, CN
中科院分区：
文献类型：
--
作者：
LI, O;HEATH, CV;COLE, CN
为了确定其产物在信使RNA的核质输出中发挥潜在作用的基因，我们分离了温度敏感的酿酒酵母菌株，并用荧光原位杂交对其进行了检测。使用地高辛标记的寡核苷酸(Dt)(50)探针，我们鉴定了当细胞移动到不允许的温度时，显示出聚(A)(+)RNA核积聚的那些。在这里，我们描述了携带RAT3-1突变(大鼠核糖核酸转移)的酵母菌株的特性和RAT3基因的克隆。在23℃的允许温度下，携带rat3-1等位基因的细胞中，只有不到10%的细胞显示出聚(A)(+)RNA的核积聚，而在37℃培养4h后，大约70%的细胞显示出聚(A)(+)RNA的核积聚。在野生型细胞中，核孔复合体(NPC)相对均匀地分布在核膜周围。RAT3-1细胞的间接免疫荧光分析和电子显微镜观察都表明，在突变细胞中，NPC聚集在NE的一个或几个区域。在15摄氏度至37摄氏度的温度下培养的突变细胞中也发现了类似的NPC聚集性。RAT3基因编码一个1157个氨基酸的蛋白质，与其他已知蛋白质没有相似之处。只有在37℃时，它才是生长所必需的。携带RAT3基因突变的细胞与携带原始RAT3-1突变的细胞非常相似；它们表现出依赖于温度的聚(A)(+)RNA的核积累，并表现出NPC的结构性聚集。Rat3p的表位标记表明，它位于核周，与RL1单抗识别的核孔蛋白共定位。我们将这种核孔素称为Rat3p/Nup133p。
To identify genes whose products play potential roles in the nucleocytoplasmic export of messenger RNA, we isolated temperature-sensitive strains of Saccharomyces cerevisiae and examined them by fluorescent in situ hybridization. With the use of a digoxigen-tagged oligo-(dT)(50) probe, we identified those that showed nuclear accumulation of poly(A)(+) RNA when cells were shifted to the nonpermissive temperature. We describe here the properties of yeast strains bearing the rat3-1 mutation (RAT - ribonucleic acid trafficking) and the cloning of the RAT3 gene. When cultured at the permissive temperature of 23 degrees C, fewer than 10% of cells carrying the rat3-1 allele showed nuclear accumulation of poly(A)(+) RNA, whereas approximately 70% showed nuclear accumulation of poly(A)(+) RNA after a shift to 37 degrees C for 4 h. In wild-type cells, nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope. Both indirect immunofluorescence analysis and electron microscopy of rat3-1 cells indicated that NPCs were clustered into one or a few regions of the NE in mutant cells. Similar NPC clustering was seen in mutant cells cultured at temperatures between 15 degrees C and 37 degrees C. The RAT3 gene encodes an 1157-amino acid protein without similarity to other known proteins. It is essential for growth only at 37 degrees C. Cells carrying a disruption of the RAT3 gene were very similar to cells carrying the original rat3-1 mutation; they showed temperature-dependent nuclear accumulation of poly(A)(+) RNA and exhibited constitutive clustering of NPCs. Epitope tagging of Rat3p demonstrated that it is located at the nuclear periphery and co-localizes with nuclear pore proteins recognized by the RL1 monoclonal antibody. We refer to this nucleoporin as Rat3p/Nup133p.