Research‚Φ–ί‚ι

The 2nd international fission yeast meeting@(2002”N7ŒŽA‹ž“sŽs ‹ž“s‘Ϋ‰ο‹cκ)
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A MUTATION IN THE GENE ENCODING A HOMOLOGUE OF HUMAN XPB/ERCC3 CAUSES A DEFECT OF mRNA EXPORT FROM THE NUCLEUS
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Fumitaka Mizuki1, Takeshi Namiki1, Hiromi Furukawa1, Yasumi Ohshima1and Tokio Tani2
(1Dept. Biol., Kyushu Univ., Fukuoka 812-8581, Japan, 2Dept. Biol. Sci., Fac. of Sci., Kumamoto Univ., Kumamoto, 860-8555, Japan)
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@Gene expression in eukaryotic cells requires selective export of spliced mRNAs from the nucleus to the cytoplasm. To identify factors involved in that process, we have isolated conditional mutants defective in mRNA export at the nonpermissive temperature, which were designated as ptr1~11(poly A+ RNA transport). Of those, the ptr8+ gene was found to encode a protein homologous with human XPB (Xeroderma pigmentosum-B) /ERCC3 (Excision repair cross complementing 3), which is a component of the transcription factor complex TFIIH. UV irradiation experiments revealed that ptr8-1 has high UV sensitivity at the semi-permissive temperature, suggesting that Ptr8p is a functional homologue for XPB/ERCC3.
@The ptr8-1 mutant accumulated poly A+ RNA in the nuclei very rapidly after shifting to the nonpermissive temperature. To verify that nuclear accumulation of poly A+ RNA in the ptr8-1 mutant is caused by inhibition of mRNA export, we carried out a pulse-labelling assay using [35S] Methionine. As a result, it was shown that protein synthesis in the ptr8-1 mutant decreased rapidly after sifting to the nonpermissive temperature, thereby demonstrating that Ptr8p plays a direct role in the mRNA export pathway. Then, we examined localization of mRNA accumulated in the nuclei at the nonpermissive temperature by a triple staining of the cells using an oligo dT probe, anti fibrillarin antibody and DAPI. We found that a majority of mRNA is accumulated in the nuclear region excluding the nucleolus in ptr8-1 at the nonpermissive temperature. To determine whether ptr8-1 has a defect in export of proteins with a leucine-rich NES at the nonpermissive temperature, we transformed ptr8-1 with the plasmid encoding a GST-SV40 NES-GFP-Pap1 NLS fusion protein and observed the cellular localization of the expressed GFP fusion protein at the nonpermissive temperature. The fusion protein was localized in both the cytoplasm and the nucleus at the nonpermissive temperature, suggesting that ptr8-1 has no defect in protein export from the nucleus. Based on these results, we propose that Ptr8p has two functions, one is defined by an UV repair defect, and the other is an essential function related to the mRNA export.