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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 and stabilizes ARE-mRNAs. (A) HuR, which bound to mRNA, was isolated from the nuclear (N) and cytoplasmic (C) fractions of each BRK cell exposed to UV light by oligo (dT)-cellulose chromatography (left). The amount of HuR in total extract (TE) of each cell is shown (middle). The fractions and TE were analyzed by immunoblotting with antibodies to β-tubulin and poly(ADP-ribose)polymerase (PARP; right). (B) The amount of c-fos, c-myc, COX-2, and GAPDH mRNA expressed in the cytoplasm of each cell was measured by quantitative real-time RT-PCR. BRK E1 and E1+E4 cells (left), 293 cells transfected with E4orf6 expression vector (middle), and HeLa cells infected with Ad dl309 and Ad dl355 (right) were used. (C) pCMVGL-ARE, which has ARE of c-fos in 3′-UTR of luciferase cDNA (left), were transfected into 293 cells with or without E4orf6 expression construct, and then the accumulation of the cytoplasmic luciferase mRNA was analyzed by Northern blot 24 h after the transfection. The quantity of 18S RNA of each cell is shown. (D) BRK cells were treated with actinomycin D and the amount of each cytoplasmic ARE-mRNA was estimated at the indicated time by quantitative real-time RT-PCR. Data are mean ± SEM of three independent experiments. (E) The distribution of ARE-mRNAs was examined in BRK cells. BRK E1+E4 (a–c), E1 (d–f), dl210-294 (g–i), and NES (−) (j–l) cells were subjected to in situ hybridization by using digoxigenin-labeled antisense oligonucleotide probes complementary to c-fos (a, d, g, and j), c-myc (b, e, h, and k), and COX-2 (c, f, i, and l) mRNAs and rhodamine-conjugated antidigoxigenin antibody. DAPI-stained nuclei are shown. (F) ARE-mRNAs associated with HuR were isolated by RIP analysis using the nuclear (N) and cytoplasmic (C) fractions of BRK cells. In, 10% input of each fraction; P, pellet; S, supernatant of immunoprecipitation.
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fig2: E4orf6 exports and stabilizes ARE-mRNAs. (A) HuR, which bound to mRNA, was isolated from the nuclear (N) and cytoplasmic (C) fractions of each BRK cell exposed to UV light by oligo (dT)-cellulose chromatography (left). The amount of HuR in total extract (TE) of each cell is shown (middle). The fractions and TE were analyzed by immunoblotting with antibodies to β-tubulin and poly(ADP-ribose)polymerase (PARP; right). (B) The amount of c-fos, c-myc, COX-2, and GAPDH mRNA expressed in the cytoplasm of each cell was measured by quantitative real-time RT-PCR. BRK E1 and E1+E4 cells (left), 293 cells transfected with E4orf6 expression vector (middle), and HeLa cells infected with Ad dl309 and Ad dl355 (right) were used. (C) pCMVGL-ARE, which has ARE of c-fos in 3′-UTR of luciferase cDNA (left), were transfected into 293 cells with or without E4orf6 expression construct, and then the accumulation of the cytoplasmic luciferase mRNA was analyzed by Northern blot 24 h after the transfection. The quantity of 18S RNA of each cell is shown. (D) BRK cells were treated with actinomycin D and the amount of each cytoplasmic ARE-mRNA was estimated at the indicated time by quantitative real-time RT-PCR. Data are mean ± SEM of three independent experiments. (E) The distribution of ARE-mRNAs was examined in BRK cells. BRK E1+E4 (a–c), E1 (d–f), dl210-294 (g–i), and NES (−) (j–l) cells were subjected to in situ hybridization by using digoxigenin-labeled antisense oligonucleotide probes complementary to c-fos (a, d, g, and j), c-myc (b, e, h, and k), and COX-2 (c, f, i, and l) mRNAs and rhodamine-conjugated antidigoxigenin antibody. DAPI-stained nuclei are shown. (F) ARE-mRNAs associated with HuR were isolated by RIP analysis using the nuclear (N) and cytoplasmic (C) fractions of BRK cells. In, 10% input of each fraction; P, pellet; S, supernatant of immunoprecipitation.

Mentions: We observed the export of HuR protein with its target mRNA in the presence of E4orf6 using in vivo UV cross-linking. The results show that HuR protein was exported with its target mRNA to the cytoplasm in BRK E1+E4 cells, whereas it existed in the nucleus of BRK E1 cells (Fig. 2 A, left). The amount of HuR in total extract of each cell was not changed by E4orf6 (Fig. 2 A, middle) and cell fractionation was confirmed by immunoblotting (Fig. 2 A, right).


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 and stabilizes ARE-mRNAs. (A) HuR, which bound to mRNA, was isolated from the nuclear (N) and cytoplasmic (C) fractions of each BRK cell exposed to UV light by oligo (dT)-cellulose chromatography (left). The amount of HuR in total extract (TE) of each cell is shown (middle). The fractions and TE were analyzed by immunoblotting with antibodies to β-tubulin and poly(ADP-ribose)polymerase (PARP; right). (B) The amount of c-fos, c-myc, COX-2, and GAPDH mRNA expressed in the cytoplasm of each cell was measured by quantitative real-time RT-PCR. BRK E1 and E1+E4 cells (left), 293 cells transfected with E4orf6 expression vector (middle), and HeLa cells infected with Ad dl309 and Ad dl355 (right) were used. (C) pCMVGL-ARE, which has ARE of c-fos in 3′-UTR of luciferase cDNA (left), were transfected into 293 cells with or without E4orf6 expression construct, and then the accumulation of the cytoplasmic luciferase mRNA was analyzed by Northern blot 24 h after the transfection. The quantity of 18S RNA of each cell is shown. (D) BRK cells were treated with actinomycin D and the amount of each cytoplasmic ARE-mRNA was estimated at the indicated time by quantitative real-time RT-PCR. Data are mean ± SEM of three independent experiments. (E) The distribution of ARE-mRNAs was examined in BRK cells. BRK E1+E4 (a–c), E1 (d–f), dl210-294 (g–i), and NES (−) (j–l) cells were subjected to in situ hybridization by using digoxigenin-labeled antisense oligonucleotide probes complementary to c-fos (a, d, g, and j), c-myc (b, e, h, and k), and COX-2 (c, f, i, and l) mRNAs and rhodamine-conjugated antidigoxigenin antibody. DAPI-stained nuclei are shown. (F) ARE-mRNAs associated with HuR were isolated by RIP analysis using the nuclear (N) and cytoplasmic (C) fractions of BRK cells. In, 10% input of each fraction; P, pellet; S, supernatant of immunoprecipitation.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: E4orf6 exports and stabilizes ARE-mRNAs. (A) HuR, which bound to mRNA, was isolated from the nuclear (N) and cytoplasmic (C) fractions of each BRK cell exposed to UV light by oligo (dT)-cellulose chromatography (left). The amount of HuR in total extract (TE) of each cell is shown (middle). The fractions and TE were analyzed by immunoblotting with antibodies to β-tubulin and poly(ADP-ribose)polymerase (PARP; right). (B) The amount of c-fos, c-myc, COX-2, and GAPDH mRNA expressed in the cytoplasm of each cell was measured by quantitative real-time RT-PCR. BRK E1 and E1+E4 cells (left), 293 cells transfected with E4orf6 expression vector (middle), and HeLa cells infected with Ad dl309 and Ad dl355 (right) were used. (C) pCMVGL-ARE, which has ARE of c-fos in 3′-UTR of luciferase cDNA (left), were transfected into 293 cells with or without E4orf6 expression construct, and then the accumulation of the cytoplasmic luciferase mRNA was analyzed by Northern blot 24 h after the transfection. The quantity of 18S RNA of each cell is shown. (D) BRK cells were treated with actinomycin D and the amount of each cytoplasmic ARE-mRNA was estimated at the indicated time by quantitative real-time RT-PCR. Data are mean ± SEM of three independent experiments. (E) The distribution of ARE-mRNAs was examined in BRK cells. BRK E1+E4 (a–c), E1 (d–f), dl210-294 (g–i), and NES (−) (j–l) cells were subjected to in situ hybridization by using digoxigenin-labeled antisense oligonucleotide probes complementary to c-fos (a, d, g, and j), c-myc (b, e, h, and k), and COX-2 (c, f, i, and l) mRNAs and rhodamine-conjugated antidigoxigenin antibody. DAPI-stained nuclei are shown. (F) ARE-mRNAs associated with HuR were isolated by RIP analysis using the nuclear (N) and cytoplasmic (C) fractions of BRK cells. In, 10% input of each fraction; P, pellet; S, supernatant of immunoprecipitation.
Mentions: We observed the export of HuR protein with its target mRNA in the presence of E4orf6 using in vivo UV cross-linking. The results show that HuR protein was exported with its target mRNA to the cytoplasm in BRK E1+E4 cells, whereas it existed in the nucleus of BRK E1 cells (Fig. 2 A, left). The amount of HuR in total extract of each cell was not changed by E4orf6 (Fig. 2 A, middle) and cell fractionation was confirmed by immunoblotting (Fig. 2 A, right).

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