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. 2005 Jul 4;170(1):15-20.
doi: 10.1083/jcb.200405112. Epub 2005 Jun 27.

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

Fumihiro Higashino  1 , Mariko Aoyagi, Akiko Takahashi, Masaho Ishino, Masato Taoka, Toshiaki Isobe, Masanobu Kobayashi, Yasunori Totsuka, Takao Kohgo, Masanobu Shindoh
Affiliations

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

Fumihiro Higashino et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
E4orf6 interacts with pp32/LANP. (A) Identification of pp32/LANP as an associated protein of E4orf6 using 293 cells transfected with expression constructs for FLAG-E4orf6 by M2 (anti-FLAG antibody) affinity column chromatography. Arrows indicate the proteins identified as FLAG-E4orf6 and pp32/LANP by MALDI-TOF/MS analysis. (B) The interaction of E4orf6 with pp32/LANP was confirmed using transformed BRK cells. The expressions of E4orf6, FLAG-E4orf6, and pp32/LANP are shown. (C) Schematic diagram of deletion mutants of E4orf6. NES, nuclear export signal; CCR, conserved cysteine-rich region; Helix, amphipathic α helix region. Bar indicates the oncodomain (top). In vitro–translated E4orf6 mutants were incubated with GST-pp32/LANP, and the associated E4orf6 mutants were confirmed by immunoblotting using the antibody to E4orf6 and HA tag (for dl1-203; bottom). (D) Subcellular localization of pp32/LANP (a and d) and E4orf6 (b and e) in mixture of BRK E1 (arrows) and E1+E4 (top) or BRK dl210-294 (bottom) cells. (c and f) Phase images are shown.
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
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.
Figure 4.
Figure 4.
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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