Shibata Satoshi, Matsuoka Yosuke, Yoneda Yoshihiro
Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Genes Cells. 2002 Apr;7(4):421-34. doi: 10.1046/j.1365-2443.2002.00525.x.
It is known that Tpr is a component of an intranuclear long filament which extends from the nuclear pore complex (NPC) into the nucleoplasm. Since the over-expression of the full-length of or some fragments of Tpr in living cells leads to the accumulation of poly(A)+ RNA within the nuclei, it is generally thought that a relationship exists between Tpr and the nuclear export of mRNA in mammalian cells. In contrast, the nuclear export of poly(A)+ RNA was not inhibited in a double deletion mutant of yeast Tpr homologues (Mlp1p and Mlp2p). Therefore, the precise function of Tpr remains unknown.
By microinjecting two types of polyclonal antibodies which are specific to Tpr into the cytoplasm of living mammalian interphase cells, we succeeded in reconstituting the Tpr-less nuclei. In the Tpr-less nuclei, the localization of the major components of the NPC, the nuclear import of SV40 T-NLS substrates and the nuclear export of HIV Rev NES-substrates were not affected. However poly(A)+ RNA accumulated in the non-snRNP splicing factor SC35-positive clusters, which became larger in size and fewer in number, compared with normal nuclei.
These results indicate that Tpr plays a critical role in the intranuclear dynamics of RNA pol II transcripts, including the processing, intranuclear transport and targeting, as well as their translocation through the NPC in mammalian cells.
已知Tpr是一种核内长丝的组成部分，该长丝从核孔复合体（NPC）延伸至核质。由于在活细胞中全长或某些Tpr片段的过表达会导致聚腺苷酸（poly(A)+）RNA在细胞核内积累，因此通常认为Tpr与哺乳动物细胞中mRNA的核输出之间存在关联。相比之下，在酵母Tpr同源物（Mlp1p和Mlp2p）的双缺失突变体中，poly(A)+ RNA的核输出并未受到抑制。因此，Tpr的确切功能仍然未知。
通过将两种对Tpr特异的多克隆抗体显微注射到活的哺乳动物间期细胞的细胞质中，我们成功重建了无Tpr的细胞核。在无Tpr的细胞核中，NPC主要成分的定位、SV40 T-NLS底物的核输入以及HIV Rev NES底物的核输出均未受影响。然而，poly(A)+ RNA在非snRNP剪接因子SC35阳性簇中积累，与正常细胞核相比，这些簇的尺寸变大且数量减少。
这些结果表明，Tpr在RNA聚合酶II转录本的核内动态过程中发挥关键作用，包括加工、核内运输和靶向，以及它们通过NPC在哺乳动物细胞中的转运。
