2001Eukaryotic mRNA ProcessingF



NUCLEOCYTOPLASMIC TRANSPORT OF FLUORESCENT mRNA IN LIVING
CELLS: ACTIVE GENE TRANSCRIPTION IS REQUIRED FOR EFFICIENT EXPORT
OF INJECTED mRNA

Toshiharu Shibuya1, Yo Ishihama2, Hisashi Tadakuma2, Takashi Funatsu2
and Tokio Tani3
1Department of Biology, Graduate school of Sciences, Kyushu University,
Fukuoka 812-8581, Japan
2Department of Physics, School of Science and Engineering, Waseda
University, 3-4-1 Okubo, Tokyo 169-8555, Japan
3Department of Biological Science, Faculty of Science, Kumamoto
University, Kumamoto 860-8555, Japan


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In eukaryotic cells, mRNA export from the nucleus to the cytoplasm is one of the important steps for gene expression. To examine the mechanism of nucleocytoplasmic transport of mRNA, we microinjected fluorescent labeled b-globin mRNA into the nuclei of HeLa and Xenopus A6 cells. The injected fluorescent mRNA moved from the nucleus to the cytoplasm with incubation time. Such relocalization of fluorescent mRNA was blocked by following treatments, (1) incubation of the injected cells at 4 C, (2) co-injection with WGA, and (3) treatment of cells with sodium azide, suggesting that migration of the injected fluorescent mRNA to the cytoplasm was proceeded via a nuclear pore-mediated and energy-required active process similar to that for the nuclear export of cellular mRNAs. In this assay system, both the m7G cap structure and polyA tail stimulated the export of mRNA from the nucleus. Interestingly, export of the injected mRNA was inhibited by treatment of cells with Actinomycin D or a-amanitin, which inhibits transcription of the genes. The injected mRNA was accumulated to form several foci in the nuclei of the drug-treated cells. Those foci were found to localize side by side with reorganized SC35 speckles in the cells. These results suggest the presence of a crosstalk between gene transcription and mRNA export, and that active gene transcription is prerequisite for nucleocytoplasmic transport of transcribed mRNAs.

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