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ABSTRACT: Recent research into mRNA maturation processes in the nucleus has identified a number of proteins involved in mRNA transcription, capping, splicing, end processing and export. Among them, the Tap-p15 heterodimer acts as an mRNA export receptor. Tap-p15 is recruited onto fully processed mRNA in the nucleus, which is ready for export to the cytoplasm, through associating with Aly or SR proteins on mRNA, or by directly associating with a constitutive transport element (CTE), an RNA element derived from type D retroviruses. mRNA containing a CTE is exported to the cytoplasm by directly associating with Tap-p15, even in the absence of Tap-recruiting proteins such as Aly or SR proteins on the mRNA. Here, we showed that the use of a CTE enhanced the expression of recombinant protein in human cell lines. The co-expression of reporter proteins and Tap-p15 also enhanced recombinant protein expression. Moreover, the use of a CTE and Tap-p15 synergistically further enhanced the recombinant protein expression. In addition to Tap-p15, several Tap-p15-recruiting proteins, including Aly and SR proteins, enhanced recombinant protein expression, albeit independently of the CTE. The incorporation of a CTE and Tap-p15-recruiting proteins into protein expression system is useful to increase recombinant protein yield in human cells.
Journal of biotechnology 05/2011; 153(3-4):86-91. · 2.88 Impact Factor
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ABSTRACT: A 50-year-old man presented with an ileocecal tumor and a large amount of ascites. Lymphoma cells obtained from the ascitic fluid were CD10(+), CD20(+), CD38(+), HLA-DR(+), BCL6(-), MUM1/IRF4(+), BCL2(+), and immunoglobulin µ/γ(+). The karyotype determined by G-banding and spectral karyotyping was 46, XY, der(3)t(1;3)(q12;p12), -4, +7, t(8;14)(q24;q32), t(12;14)(q24;q32), der(17)t(4;17)(q21;p11). Fluorescence in situ hybridization disclosed that 93% of interphase cells were positive for the c-MYC and immunoglobulin heavy chain gene fusion. The patient was treated with intensive chemo-immunotherapy, resulting in a complete response. The t(8;14)-t(12;14) double-hit may have generated molecular abnormalities analogous to those of a previously cloned three-way translocation t(8;12;14).
Internal Medicine 01/2011; 50(21):2659-62. · 0.94 Impact Factor
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Tomohiro Yamazaki,
Naoko Fujiwara,
Hiroko Yukinaga,
Miki Ebisuya,
Takuya Shiki,
Tomoya Kurihara,
Noriyuki Kioka,
Taiho Kambe,
Masaya Nagao,
Eisuke Nishida,
Seiji Masuda
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ABSTRACT: Nuclear export of mRNA is an essential process for eukaryotic gene expression. The TREX complex couples gene expression from transcription and splicing to mRNA export. Sub2, a core component of the TREX complex in yeast, has diversified in humans to two closely related RNA helicases, UAP56 and URH49. Here, we show that URH49 forms a novel URH49-CIP29 complex, termed the AREX (alternative mRNA export) complex, whereas UAP56 forms the human TREX complex. The mRNAs regulated by these helicases are different at the genome-wide level. The two sets of target mRNAs contain distinct subsets of key mitotic regulators. Consistent with their target mRNAs, depletion of UAP56 causes mitotic delay and sister chromatid cohesion defects, whereas depletion of URH49 causes chromosome arm resolution defects and failure of cytokinesis. In addition, depletion of the other human TREX components or CIP29 causes mitotic defects similar to those observed in UAP56- or URH49-depleted cells, respectively. Taken together, the two closely related RNA helicases have evolved to form distinct mRNA export machineries, which regulate mitosis at different steps.
Molecular biology of the cell 08/2010; 21(16):2953-65. · 5.98 Impact Factor
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ABSTRACT: Screening of mRNA export factors in Saccharomyces cerevisiae and Drosophila melanogaster has identified a number of mRNA processing factors involved in multiple mRNA processing steps. However, only limited information is available on human cells. Here we established a screening system searching for mRNA processing factors in human cells by combining the luciferase reporter system and fluorescence in situ hybridization, which evaluates the nuclear/cytoplasmic distribution of bulk poly(A)+ RNA. This system makes it possible to search for the compounds affecting mRNA processing from the various resources.
Bioscience Biotechnology and Biochemistry 01/2010; 74(7):1512-6. · 1.28 Impact Factor
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Ayako Fukunaka,
Tomoyuki Suzuki,
Yayoi Kurokawa, Tomohiro Yamazaki,
Naoko Fujiwara,
Kaori Ishihara,
Hitoshi Migaki,
Katsuzumi Okumura,
Seiji Masuda,
Yuko Yamaguchi-Iwai,
Masaya Nagao,
Taiho Kambe
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ABSTRACT: The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.
Journal of Biological Chemistry 09/2009; 284(45):30798-806. · 4.77 Impact Factor
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ABSTRACT: The SLC39A family of zinc transporters can be divided into four subfamilies (I, II, LIV-1, and gufA) in vertebrates, but studies of their functions have been restricted exclusively to members of subfamilies II and LIV-1. In this study, we characterized SLC39A9 (ZIP9), the only member of subfamily I in vertebrates. Confocal microscopy demonstrated that transiently expressed, HA-tagged human ZIP9 (hZIP9-HA) was localized to the trans-Golgi network regardless of zinc status. Disruption of the ZIP9 gene in DT40 cells did not change the growth rate, sensitivity to high zinc and manganese concentrations during long-term culture, or cellular zinc status after short-term incubation with zinc. The alkaline phosphatase activity of ZIP9(-/-) cells did not change in cells cultured in medium containing normal zinc levels. In contrast, the activity of this enzyme decreased in wild-type cells cultured in zinc deficient medium but less so in ZIP9(-/-) cells under these conditions. Stable over-expression of hZIP9-HA moderately decreased alkaline phophatase activity. These results suggest that ZIP9 functions to regulate zinc homeostasis in the secretory pathway without significantly altering cytosolic zinc homeostasis.
Bioscience Biotechnology and Biochemistry 06/2009; 73(5):1142-8. · 1.28 Impact Factor
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ABSTRACT: Zinc transporters play important roles in a wide range of biochemical processes. Here we report an important function of ZnT5/ZnT6 hetero-oligomeric complexes in the secretory pathway. The activity of human tissue-nonspecific alkaline phosphatase (ALP) expressed in ZnT5(-)ZnT7(-/-) cells was significantly reduced compared with that expressed in wild-type cells as in the case of endogenous chicken tissue-nonspecific ALP activity. The inactive human tissue-nonspecific ALP in ZnT5(-)ZnT7(-/-) cells was degraded by proteasome-mediated degradation without being trafficked to the plasma membrane. ZnT5(-)ZnT7(-/-) cells showed exacerbation of the unfolded protein response as did the wild-type cells cultured under a zinc-deficient condition, revealing that both complexes play a role in homeostatic maintenance of secretory pathway function. Furthermore, we showed that expression of ZnT5 mRNA was up-regulated by the endoplasmic reticulum stress in various cell lines. The up-regulation of the hZnT5 transcript was mediated by transcription factor XBP1 through the TGACGTGG sequence in the hZnT5 promoter, and this sequence was highly conserved in the ZnT5 genes of mouse and chicken. These results suggest that zinc transport into the secretory pathway is strictly regulated for the homeostatic maintenance of secretory pathway function in vertebrate cells.
Journal of Biological Chemistry 07/2006; 281(26):17743-50. · 4.77 Impact Factor
