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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, E1B-55kD, pp32/LANP, and HuR are associated with c-fos mRNA. (A) c-fos mRNA coprecipitates in the immunoprecipitation of HuR, pp32/LANP, E4orf6, and E1B-55kD from the cytoplasmic and nuclear fractions of each BRK cell. Mouse IgG (mIgG) was used as a control for the antibodies. (B) Coprecipitated HuR protein in the precipitates of pp32/LANP-specific antibody was immunoblotted to visualize HuR-pp32/LANP binding using 293 cells. The expressions of transfected HuR and pp32/LANP are shown. (C) The same transfected cells were used to observe in vivo binding between E4orf6 and HuR using the indicated antibodies. On the bottom are the expression of E4orf6 and HuR (left). In vitro binding was confirmed using GST-HuR and in vitro translated (IVT) E4orf6 (right). (D) The interaction between E4orf6 and pp32/LANP was analyzed in the presence of RNaseA using 293 cells transfected with indicated plasmids. The expression of pp32/LANP and FLAG-E4orf6 are shown.
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fig3: E4orf6, E1B-55kD, pp32/LANP, and HuR are associated with c-fos mRNA. (A) c-fos mRNA coprecipitates in the immunoprecipitation of HuR, pp32/LANP, E4orf6, and E1B-55kD from the cytoplasmic and nuclear fractions of each BRK cell. Mouse IgG (mIgG) was used as a control for the antibodies. (B) Coprecipitated HuR protein in the precipitates of pp32/LANP-specific antibody was immunoblotted to visualize HuR-pp32/LANP binding using 293 cells. The expressions of transfected HuR and pp32/LANP are shown. (C) The same transfected cells were used to observe in vivo binding between E4orf6 and HuR using the indicated antibodies. On the bottom are the expression of E4orf6 and HuR (left). In vitro binding was confirmed using GST-HuR and in vitro translated (IVT) E4orf6 (right). (D) The interaction between E4orf6 and pp32/LANP was analyzed in the presence of RNaseA using 293 cells transfected with indicated plasmids. The expression of pp32/LANP and FLAG-E4orf6 are shown.

Mentions: We performed an RIP assay to observe the interaction between the protein complex and c-fos mRNA. In BRK E1 cells, c-fos mRNA was coprecipitated with HuR and pp32/LANP proteins only in the nuclear fraction. On the other hand, it was coprecipitated with HuR, pp32/LANP, E4orf6, and E1B-55kD in both the nuclear and cytoplasmic fractions of BRK E1+E4 cells (Fig. 3 A). This suggests that these proteins are associated with c-fos mRNA and are all exported to the cytoplasm when cells express E4orf6. Interestingly, E1B-55kD associated with c-fos mRNA in BRK E1+E4 cells, but not in BRK E1 cells. This finding indicates that E4orf6 is necessary for E1B-55kD to associate with c-fos mRNA. This is the first evidence showing that E4orf6 transports mRNA by association with its target mRNA.


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, E1B-55kD, pp32/LANP, and HuR are associated with c-fos mRNA. (A) c-fos mRNA coprecipitates in the immunoprecipitation of HuR, pp32/LANP, E4orf6, and E1B-55kD from the cytoplasmic and nuclear fractions of each BRK cell. Mouse IgG (mIgG) was used as a control for the antibodies. (B) Coprecipitated HuR protein in the precipitates of pp32/LANP-specific antibody was immunoblotted to visualize HuR-pp32/LANP binding using 293 cells. The expressions of transfected HuR and pp32/LANP are shown. (C) The same transfected cells were used to observe in vivo binding between E4orf6 and HuR using the indicated antibodies. On the bottom are the expression of E4orf6 and HuR (left). In vitro binding was confirmed using GST-HuR and in vitro translated (IVT) E4orf6 (right). (D) The interaction between E4orf6 and pp32/LANP was analyzed in the presence of RNaseA using 293 cells transfected with indicated plasmids. The expression of pp32/LANP and FLAG-E4orf6 are shown.
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Related In: Results  -  Collection

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

fig3: E4orf6, E1B-55kD, pp32/LANP, and HuR are associated with c-fos mRNA. (A) c-fos mRNA coprecipitates in the immunoprecipitation of HuR, pp32/LANP, E4orf6, and E1B-55kD from the cytoplasmic and nuclear fractions of each BRK cell. Mouse IgG (mIgG) was used as a control for the antibodies. (B) Coprecipitated HuR protein in the precipitates of pp32/LANP-specific antibody was immunoblotted to visualize HuR-pp32/LANP binding using 293 cells. The expressions of transfected HuR and pp32/LANP are shown. (C) The same transfected cells were used to observe in vivo binding between E4orf6 and HuR using the indicated antibodies. On the bottom are the expression of E4orf6 and HuR (left). In vitro binding was confirmed using GST-HuR and in vitro translated (IVT) E4orf6 (right). (D) The interaction between E4orf6 and pp32/LANP was analyzed in the presence of RNaseA using 293 cells transfected with indicated plasmids. The expression of pp32/LANP and FLAG-E4orf6 are shown.
Mentions: We performed an RIP assay to observe the interaction between the protein complex and c-fos mRNA. In BRK E1 cells, c-fos mRNA was coprecipitated with HuR and pp32/LANP proteins only in the nuclear fraction. On the other hand, it was coprecipitated with HuR, pp32/LANP, E4orf6, and E1B-55kD in both the nuclear and cytoplasmic fractions of BRK E1+E4 cells (Fig. 3 A). This suggests that these proteins are associated with c-fos mRNA and are all exported to the cytoplasm when cells express E4orf6. Interestingly, E1B-55kD associated with c-fos mRNA in BRK E1+E4 cells, but not in BRK E1 cells. This finding indicates that E4orf6 is necessary for E1B-55kD to associate with c-fos mRNA. This is the first evidence showing that E4orf6 transports mRNA by association with its target mRNA.

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