Azad A K, Tani T, Shiki N, Tsuneyoshi S, Urushiyama S, Ohshima Y
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Mol Biol Cell. 1997 May;8(5):825-41. doi: 10.1091/mbc.8.5.825.
Nucleocytoplasmic transport of mRNA is essential for eukaryotic gene expression. However, how mRNA is exported from the nucleus is mostly unknown. To elucidate the mechanisms of mRNA transport, we took a genetic approach to identify genes, the products of which play a role in that process. From about 1000 temperature -sensitive (ts- or cs-) mutants, we identified five ts- mutants that are defective in poly(A)+ RNA transport by using a situ hybridization with an oligo(dT)50 as a probe. These mutants accumulate poly(A)+ RNA in the nuclei when shifted to a nonpermissive temperature. All five mutations are tightly linked to the ts- growth defects, are recessive, and fall into four different groups designated as ptr 1-4 (poly(A)+ RNA transport). Interestingly, each group of mutants has a differential localization pattern of poly(A)+ RNA in the nuclei at the nonpermissive temperature, suggesting that they have defects at different steps of the mRNA transport pathway. Localization of a nucleoplasmin-green fluorescent protein fusion suggests that ptr2 and ptr3 have defects also in nuclear protein import. Among the isolated mutants, only ptr2 showed a defect in pre-mRNA splicing. We cloned the ptr2+ and ptr3+ genes and found that they encode Schizosaccharomyces pombe homologues of the mammalian RCC1, a guanine nucleotide exchange factor for RAN/TC4, and the ubiquitin-activating enzyme E1 involved in ubiquitin conjugation, respectively. The ptr3+ gene is essential for cell viability, and Ptr3p tagged with green fluorescent protein was localized in both the nucleus and the cytoplasm. This is the first report suggesting that the ubiquitin system plays a role in mRNA export.
mRNA的核质运输对于真核基因表达至关重要。然而，mRNA如何从细胞核输出大多仍不清楚。为了阐明mRNA运输的机制，我们采用遗传学方法来鉴定那些其产物在该过程中起作用的基因。从大约1000个温度敏感（ts-或cs-）突变体中，我们通过使用寡聚（dT）50作为探针的原位杂交，鉴定出五个在多聚（A）+RNA运输方面有缺陷的ts-突变体。当转移到非允许温度时，这些突变体在细胞核中积累多聚（A）+RNA。所有五个突变都与ts-生长缺陷紧密连锁，是隐性的，并分为四个不同的组，命名为ptr 1 - 4（多聚（A）+RNA运输）。有趣的是，每组突变体在非允许温度下细胞核中多聚（A）+RNA具有不同的定位模式，这表明它们在mRNA运输途径的不同步骤存在缺陷。核纤层蛋白 - 绿色荧光蛋白融合蛋白的定位表明，ptr2和ptr3在核蛋白输入方面也有缺陷。在分离出的突变体中，只有ptr2在前体mRNA剪接方面表现出缺陷。我们克隆了ptr2 +和ptr3 +基因，发现它们分别编码哺乳动物RCC1（一种RAN/TC4的鸟嘌呤核苷酸交换因子）和参与泛素缀合作用的泛素激活酶E1的粟酒裂殖酵母同源物。ptr3 +基因对于细胞活力是必需的，并且用绿色荧光蛋白标记的Ptr3p定位于细胞核和细胞质中。这是首次表明泛素系统在mRNA输出中起作用的报告。
