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Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism.

Higashino F, Aoyagi M, Takahashi A, Ishino M, Taoka M, Isobe T, Kobayashi M, Totsuka Y, Kohgo T, Shindoh M - J. Cell Biol. (2005)

Bottom Line: We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells.The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA.Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Sapporo 060-8586, Japan. fhigashi@den.hokudai.ac.jp

ABSTRACT
E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

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E4orf6 exports ARE-mRNA in a CRM1-independent manner. (A) Coprecipitated CRM1 in the precipitates of pp32/LANP-specific antibody was confirmed using 293 cells as described in Fig. 3 B. The expressions of CRM1, pp32/LANP, and E4orf6 are shown in the bottom three panels. (B). The quantity of cytoplasmic ARE-mRNAs was measured by quantitative real-time RT-PCR using heat-shocked (45°C, 1 h) BRK E1 (E1 HS) and BRK E1+E4 (E1+E4) cells treated with (+) or without (−) LMB. Data are mean ± SEM of three independent experiments. (C) The effect of LMB observed in B was confirmed by RIP analysis using the same cells. Mouse IgG (mIgG) was used as a control. (D) The effect of LMB for the export of c-fos mRNA was observed by in situ hybridization using BRK cells as described in Fig. 2 E. (E) HeLa cells were either treated with heat shock or transfected with E4orf6 expression vector. The cytoplasmic fraction of each cell was immunoblotted with each antibody in the presence or absence of LMB. (F) Subcellular localization of E4orf6 in BRK E1+E4 cells in the presence or absence of LMB.
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fig4: E4orf6 exports ARE-mRNA in a CRM1-independent manner. (A) Coprecipitated CRM1 in the precipitates of pp32/LANP-specific antibody was confirmed using 293 cells as described in Fig. 3 B. The expressions of CRM1, pp32/LANP, and E4orf6 are shown in the bottom three panels. (B). The quantity of cytoplasmic ARE-mRNAs was measured by quantitative real-time RT-PCR using heat-shocked (45°C, 1 h) BRK E1 (E1 HS) and BRK E1+E4 (E1+E4) cells treated with (+) or without (−) LMB. Data are mean ± SEM of three independent experiments. (C) The effect of LMB observed in B was confirmed by RIP analysis using the same cells. Mouse IgG (mIgG) was used as a control. (D) The effect of LMB for the export of c-fos mRNA was observed by in situ hybridization using BRK cells as described in Fig. 2 E. (E) HeLa cells were either treated with heat shock or transfected with E4orf6 expression vector. The cytoplasmic fraction of each cell was immunoblotted with each antibody in the presence or absence of LMB. (F) Subcellular localization of E4orf6 in BRK E1+E4 cells in the presence or absence of LMB.

Mentions: It is known that CRM1 binds to pp32/LANP and that ARE-mRNAs are exported to the cytoplasm in a CRM1-dependent manner when cells are stimulated by heat shock or serum (Brennan et al., 2000; Gallouzi et al., 2001). To observe the influence of E4orf6 on pp32/LANP–CRM1 interaction, we examined the binding intensity of both proteins. Because the quantity of coprecipitated CRM1 with pp32/LANP was almost the same between cells with and without E4orf6 expression (Fig. 4 A), E4orf6 does not disturb the binding of these proteins.


Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism.

Higashino F, Aoyagi M, Takahashi A, Ishino M, Taoka M, Isobe T, Kobayashi M, Totsuka Y, Kohgo T, Shindoh M - J. Cell Biol. (2005)

E4orf6 exports ARE-mRNA in a CRM1-independent manner. (A) Coprecipitated CRM1 in the precipitates of pp32/LANP-specific antibody was confirmed using 293 cells as described in Fig. 3 B. The expressions of CRM1, pp32/LANP, and E4orf6 are shown in the bottom three panels. (B). The quantity of cytoplasmic ARE-mRNAs was measured by quantitative real-time RT-PCR using heat-shocked (45°C, 1 h) BRK E1 (E1 HS) and BRK E1+E4 (E1+E4) cells treated with (+) or without (−) LMB. Data are mean ± SEM of three independent experiments. (C) The effect of LMB observed in B was confirmed by RIP analysis using the same cells. Mouse IgG (mIgG) was used as a control. (D) The effect of LMB for the export of c-fos mRNA was observed by in situ hybridization using BRK cells as described in Fig. 2 E. (E) HeLa cells were either treated with heat shock or transfected with E4orf6 expression vector. The cytoplasmic fraction of each cell was immunoblotted with each antibody in the presence or absence of LMB. (F) Subcellular localization of E4orf6 in BRK E1+E4 cells in the presence or absence of LMB.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171388&req=5

fig4: E4orf6 exports ARE-mRNA in a CRM1-independent manner. (A) Coprecipitated CRM1 in the precipitates of pp32/LANP-specific antibody was confirmed using 293 cells as described in Fig. 3 B. The expressions of CRM1, pp32/LANP, and E4orf6 are shown in the bottom three panels. (B). The quantity of cytoplasmic ARE-mRNAs was measured by quantitative real-time RT-PCR using heat-shocked (45°C, 1 h) BRK E1 (E1 HS) and BRK E1+E4 (E1+E4) cells treated with (+) or without (−) LMB. Data are mean ± SEM of three independent experiments. (C) The effect of LMB observed in B was confirmed by RIP analysis using the same cells. Mouse IgG (mIgG) was used as a control. (D) The effect of LMB for the export of c-fos mRNA was observed by in situ hybridization using BRK cells as described in Fig. 2 E. (E) HeLa cells were either treated with heat shock or transfected with E4orf6 expression vector. The cytoplasmic fraction of each cell was immunoblotted with each antibody in the presence or absence of LMB. (F) Subcellular localization of E4orf6 in BRK E1+E4 cells in the presence or absence of LMB.
Mentions: It is known that CRM1 binds to pp32/LANP and that ARE-mRNAs are exported to the cytoplasm in a CRM1-dependent manner when cells are stimulated by heat shock or serum (Brennan et al., 2000; Gallouzi et al., 2001). To observe the influence of E4orf6 on pp32/LANP–CRM1 interaction, we examined the binding intensity of both proteins. Because the quantity of coprecipitated CRM1 with pp32/LANP was almost the same between cells with and without E4orf6 expression (Fig. 4 A), E4orf6 does not disturb the binding of these proteins.

Bottom Line: We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells.The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA.Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Pathobiological Science, Hokkaido University Graduate School of Dental Medicine, Sapporo 060-8586, Japan. fhigashi@den.hokudai.ac.jp

ABSTRACT
E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.

Show MeSH
Related in: MedlinePlus