The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels - PubMed (original) (raw)

The human papillomavirus type 16 negative regulatory RNA element interacts with three proteins that act at different posttranscriptional levels

M D Koffa et al. Proc Natl Acad Sci U S A. 2000.

Abstract

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3' untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.

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Figures

Figure 1

Figure 1

Analysis of keratin 10 expression in W12 cells. Cells were untreated (lane 1), or treated with 16 nM PMA for 5 days (lane 2) and 10 days (lane 3), increased Ca2+ and serum concentration for 5 days (lane 4) or 10 days (lane 5), or suspended in methylcellulose for 1 day (lane 6), 2 days (lane 7), or 8 days (lane 8). Total extracts (50 μg) were used and Western blot analysis was performed with the anti-K10 Ck 8.60 antibody.

Figure 2

Figure 2

The NRE RNA binds cellular proteins. EMSA with HeLa (lanes 1 and 4), undifferentiated (−, lanes 2 and 5) and differentiating (+, lanes 3 and 6) W12 cell nuclear (lanes 1–3) and cytoplasmic (lanes 4–6) extracts. A 32P-labeled _in vitro_-transcribed sense probe, homologous to the NRE (NRE RNA) was used. Arrows indicate the complexes formed.

Figure 3

Figure 3

U2AF65 interacts with the HPV16 NRE RNA. EMSA with (A) the NRE RNA probe or (B) a probe homologous to the 3′ half of the NRE (3′ probe), incubated with HeLa nuclear extract with no Ab (lane 1) or in the presence of an Ab against hnRNPA1 (lane 2), U2AF65 (lane 3), and hnRNPK (lane 4). (C) His-tagged U2AF65 was UV cross-linked to the B2P2 probe (lane 3), the NRE probe (lane 2), and the 5′ half of the NRE (5′ probe) (lane 1). Arrows indicate the complexes formed.

Figure 4

Figure 4

CstF-64 interacts with the HPV16 NRE RNA. (A) EMSA with the NRE RNA probe incubated with HeLa cell nuclear extract in the absence (lane 1) or presence (lane 2) of a mAb against CstF-64 (α64k). (B) EMSA with the NRE probe incubated with 70 μM of GST-64RBD in the absence (lane 2) or presence of the α64k mAb (lane 3). As a control, 1 μg of GST protein was incubated with the NRE probe (lane 1). Arrows indicate the complexes formed. (C) Purified CstF from HeLa cells was UV cross-linked to an antisense (a/s) NRE probe (lane 1) and to the NRE (lane 2).

Figure 5

Figure 5

HuR binds the HPV16 NRE RNA. (A) EMSA with the NRE RNA probe incubated with HeLa cell nuclear (lanes 1 and 2) or cytoplasmic extract (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of a mAb against HuR (19F12). The arrow indicates the supershift of the bound RNA induced by the mAb. (B) EMSA with 10 μM GST-HuR incubated with the NRE probe in the absence (lane 2) or presence (lane 3) of the HuR mAb, or with an antisense (a/s) NRE probe (lane 1). (C) GST-HuR protein (10 μM) was UV cross-linked to the NRE RNA (lane 1) and an antisense (a/s) NRE probe (lane 2). As a control, GST protein was UV cross-linked to the NRE (lane 3).

Figure 6

Figure 6

Competition between CstF-64 and HuR for NRE RNA binding. EMSA with the NRE RNA probe incubated with 70 μM of GST-64RBD in the absence (lane 1) or presence of increased concentrations of GST-HuR: 1 μM (lane 2), 5 μM (lane 3), and 10 μM (lane 4). In lane 5, the NRE was incubated with 10 μM GST-HuR, alone. The arrow indicates the complex formed between GST-64RBD and NRE.

Figure 7

Figure 7

Western blot analysis of U2AF65, CstF-64, and HuR expression in HeLa cells, and undifferentiated (−) and differentiating (+) W12 cells. (A) Nuclear extract (10 μg) was used for the detection of U2AF65 with the MC3 mAb, and CstF with the α64k mAb. (B) Nuclear and cytoplasmic extracts equivalent to 105 cells, or 50 μg of total extracts were used for the detection of HuR with the 19F12 mAb. (C) Densitometric quantification of three independent experiments was performed and the differences found between undifferentiated and differentiated W12 cells are shown with standard deviations from the means.

Figure 8

Figure 8

The NRE-containing transcripts are confined in the nucleus. Northern blot analysis of nuclear (N) and cytoplasmic (cyt) poly(A)+ RNA extracted from (A) undifferentiated W12 cells with an L1-specific riboprobe; the arrows indicate the presence of two L1-containing transcripts, or (B) from HeLa cells transfected with pCAT445 (lanes 1 and 2), or pCAT227 (lanes 3 and 4) by using a CAT-specific riboprobe. Blots were stripped and rehybridized with probes for γ-actin and U6 RNA.

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