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High-risk human papillomaviruses (HPVs) encode two viral oncoproteins, E6 and E7, from a single bicistronic pre-mRNA containing three exons and two introns. Retention of intron 1 in the E6 coding region is essential for production of the full-length E6 oncoprotein. However, splicing of intron 1 is extremely efficient in cervical cancer cells, leading to the production of a spliced transcript, E6*I, of E6. Here, we investigated whether this splicing of intron 1 might benefit E7 production. Using RNA interference as a tool, we targeted the intron 1 region using small interfering RNAs (siRNAs) in HPV-positive cell lines. At an effective low dose, the siRNAs specifically suppressed E6 expression but not E7 expression, as demonstrated by the stabilization of p53. However, at high doses the HPV18 intron 1-specific siRNA substantially and specifically reduced the level of the 18E6*I mRNA lacking the intron region in HeLa cells, implying its nuclear silencing on the pre-mRNA before RNA splicing. Two other siRNAs targeting the exon 2 regions of HPV16 and -18, which encode the E7oncoprotein, reduced the E6*I mRNAs to a remarkable extent and preferentially suppressed expression of E7, leading to accumulation of hypophosphorylated p105Rb and cell cycle arrest, indicating that the majority of E7 proteins are the translational products of E6*I mRNAs. This was confirmed by transient transfection in 293 cells: E7 could be translated only from the E7 open reading frame (ORF) on E6*I mRNA in a distance-dependent matter of upstream E6*I ORF by translation reinitiation. The data thus provide direct evidence that the E6*I mRNAs of high-risk HPVs are responsible for E7 production.
Human papillomavirus (HPV) infection of epidermal or mucosal epithelial cells causes benign and sometimes malignant neoplasms. Certain HPVs, such as HPV16, -18, -31, and -45, are detected frequently in anogenital cancers, particularly cancer of the cervix and anus, and are thus considered to be high risk or oncogenic (22, 42). Among the high-risk HPVs, HPV16 and -18 cause >90% of cervical cancers (22) and >20% of oral cancers (35). High-risk HPVs encode two potent viral oncoproteins, E6 and E7, that mediate, respectively, degradation of the cellular proteins p53 and retinoblastoma protein (pRb), two tumor suppressor proteins that are essential for cell cycle control (21).
In both HPV16 and HPV18, E6 and E7 are transcribed as a single bicistronic E6E7 transcript using a common promoter and a common early polyadenylation site (1). Promoter p97, upstream of the HPV16 E6 (16E6) open reading frame (ORF), and promoter p105, upstream of the HPV18 E6 (18E6) ORF, are responsible for the initiation of transcription in each virus genome. The bicistronic E6E7 pre-mRNAs of HPV16 and HPV18 contain three exons and two introns. One of the two introns, intron 1 (the cap-proximal intron), is positioned in the E6 ORF in both the HPV16 and HPV18 E6E7 pre-mRNAs (Fig. 1A and B). Conceivably, intron 1 removal by RNA splicing would disrupt the E6 ORF and prevent full-length E6 from being expressed. How a bicistronic E6E7 pre-mRNA escapes intron 1 splicing to encode an oncogenic E6 protein remains largely unexplored. Although the intron 1 of HPV18 E6E7 (18E6E7) pre-mRNA has one 5′ splice site (5′ ss) and one 3′ ss, the intron 1 of HPV16 E6E7 (16E6E7) pre-mRNA contains one 5′ ss and three alternative 3′ ss (Fig. 1A and B). Various studies in our laboratory and others have demonstrated that splicing of intron 1 in the 16E6E7 pre-mRNA is highly efficient, and the majority of the transcripts in cancer tissues and cervical cancer cell lines are E6*I, a spliced product without intron 1 from the nucleotide (nt) 226 5′ ss to the nt 409 3′ ss (3, 9, 31, 41).
Why then is efficient splicing of intron 1 in HPV16 or HPV18 E6E7 pre-mRNA needed for viral gene expression, since splicing harms E6 expression? It has been proposed that splicing of intron 1 in 16E6E7 pre-mRNA might benefit E7 expression (29, 32), because the space between the termination of E6 translation and the reinitiation of E7 translation in an intron 1-containing 16E6E7 mRNA is limited, with only 2 nt between them. Splicing of intron 1 creates a frameshift, and the resulting E6*I mRNA obtains a pretermination codon immediately downstream of the splice junction and, accordingly, creates enough space for translation termination and reinitiation by the ribosome (18). Other studies using in vitro translation and a vaccinia virus expression vector that bypasses nuclear transcription and splicing events suggested that translation of the HPV16 oncoprotein E7 from the bicistronic mRNA is independent of intron 1 splicing, and a leaky scanning mechanism was proposed for translation of HPV16 E7 from intron 1-containing E6E7 mRNAs (33, 34). However, recent work in our laboratory using transient transfection assays demonstrated that splicing of intron 1 in 16E6E7 pre-mRNA does enhance E7 translation. Mutation of the intron 1 5′ ss or enlarging the size of exon 1 of the 16E6E7 pre-mRNA prevents RNA splicing and significantly decreases E7 expression, implying that E7 might be translated from the spliced isoforms of 16E6E7 mRNAs (41). In this report, we took RNA splicing of bicistronic E6E7 pre-mRNAs into consideration to design intron 1- or exon 2-specific small interfering RNAs (siRNAs) for RNA interference (RNAi), a technique that is emerging as a powerful tool to control gene expression (13). We used this approach to dissect viral oncogene expression and demonstrate for the first time with cervical cancer cell lines that splicing of intron 1 from the E6 coding region is essential for the production of the viral oncoprotein E7, whereas a bicistronic mRNA retaining the E6 intron translates only the viral oncoprotein E6. In addition, we also demonstrate that siRNAs can target pre-mRNAs in the nuclei of HeLa cells.
Synthetic double-stranded siRNA 198 (5′-GCACACACGUAGACAUUCGdTdT-3′, where dT designates deoxyribosylthymine) and siRNA 209 (5′-UCCAUAUGCUGUAUGUGAUdTdT-3′) were designed to target the HPV16 E7 coding region at nt 773 to 791 in exon 2 and the E6 coding region at nt 277 to 298 in intron 1 of the 16E6E7 bicistronic transcripts, respectively. Synthetic siRNA 219 (5′-CUCUGUGUAUGGAGACACAdTdT-3′) and siRNA 220 (5′-UGGAGUUAAUCAUCAACAUdTdT-3′) target the HPV18 E6 coding region at nt 353 to 371 in intron 1 and the E7 coding region at nt 715 to 733 in exon 2 of the HPV18 (18E6E7) bicistronic transcripts, respectively. A published 16E6E7 siRNA 210 (5′-GAAUGUGUGUACUGCAAGCdTdT-3′) that targets the 16E6E7 coding region at nt 188 to 206 in exon 1 was used in some experiments as a positive control (40). All siRNAs were synthesized and preannealed by Dharmacon, Inc. (Lafayette, CO). As a negative control, a nonspecific (NS) siRNA with 52% G+C content (catalogue no. D-001206-08-20) and a green fluorescent protein (GFP) siRNA (catalogue no. D-001300-01-20) were also obtained from Dharmacon.
The CaSki and SiHa cell lines are HPV16-positive human cervical carcinoma cells. HeLa cells are HPV18-positive human cervical carcinoma cells. All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. Each synthetic siRNA described above was transfected into CaSki cells, SiHa cells, or HeLa cells with Oligofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. The concentrations of the siRNAs were as labeled in each figure. To prepare protein and RNA samples at the same time, 2 × 106 cells were seeded in duplicate or triplicate 10-cm plates and grown with 10 ml of DMEM overnight before being used for transfection. The transfected cells were maintained in DMEM containing 10% fetal bovine serum at 37°C under a 5% CO2 atmosphere. At the scheduled time points after transfection, total cell protein lysates were prepared by the addition of 2× sodium dodecyl sulfate (SDS) loading buffer to the cells; the cells were then subjected to ultrasonification multiple times. Protein samples were denatured by being boiled for 2 to 3 min, separated by NuPAGE Bis-Tris gel electrophoresis (Invitrogen), transferred onto a nitrocellulose membrane, and blotted with different antibodies, including anti-HPV16 E7 (SC-6981; Santa Cruz Biotechnology, Santa Cruz, CA), anti-HPV18 E7 (SC-1590; Santa Cruz Biotechnology), anti-p21 (6B6; BD PharMingen, San Diego, CA), anti-p53 (Ab-6; Oncogene, Cambridge, MA), anti-cyclin A (SC-751; Santa Cruz Biotechnology), anti-pRb (G3-245; BD PharMingen), and anti-β tubulin (5H1; BD PharMingen).
Total-cell RNA was prepared from cervical cancer cells with or without siRNA treatment using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. A 16E6E7-specific antisense RNA probe was synthesized from a CaSki HPV16 DNA template by in vitro transcription and used for detection of the E6E7 RNAs as previously described (41). An 18E6E7-specific antisense RNA probe (targeting nt 351 to 850 at the intron 1-exon 2 junction region as described) (Fig. (Fig.1B)1B) was synthesized from a HeLa HPV18 DNA template by in vitro transcription. An antisense cyclophilin probe was prepared from a human cyclophilin A template (Ambion) and used as an internal sample loading control. A human cell cycle (hCC-2) multiprobe template set and RNase protection assay (RPA) kit were purchased from BD PharMingen. Approximately 30 μg of total-cell RNA, 105 cpm of E6E7 or cyclophilin A probe, or 3.9 × 106 cpm of hCC-2 multiprobes was used in each assay according to the manufacturer's instructions.
RNA splicing of 16E6E7 and 18E6E7 pre-mRNAs in total-cell RNA was also examined by RT-PCR using the primer pairs Pr106 (5′-GTTTCAGGACCCACAGGAGC-3′) and Pr562 (5′-GTACTCACCCC/TGATTACAGCTGGGTTTC-3′) for HPV16 and Pr121 (5′-ATCCAACACGGCGACCCTAC-3′) and Pr850 (5′-CTGGAATGCTCGAAGGTC-3′) for HPV18. To detect 16E6E7 and 18E6E7 bicistronic mRNAs retaining the E6 intron, an intron-specific primer was used for primer pairing and RT-PCR analysis as shown in Fig. 1A and B.
Cell cycle distribution was determined by flow cytometry. CaSki or HeLa cells (5 × 105) were seeded in triplicate in 6-cm petri dishes with 5 ml DMEM overnight before transfection. Transfection of each siRNA was then performed as detailed above. Cells were trypsinized 24, 48, or 72 h after siRNA transfection, washed twice with 1× cold phosphate-buffered saline at low speed, resuspended in 1 ml of Vindelov's propidium iodide buffer (37), and analyzed with a CYAN MLE cytometer (Dako-Cytomation, Fort Collins, CO). The cells were excited at 488 nm, and the fluorescence was collected on the FL2 channel using a 575/25 bandpass filter. Data were collected on a linear scale, and pulse width was used to eliminate doublets. Ten thousand events were collected per sample, and data analysis was performed using Modfit LT software (Verity Software House, Topsham, ME).
Cell telomerase activity was analyzed with a TRAPeze Gel-Based Telomerase Detection kit (Chemicon International, Inc., Temecula, CA) according to the manufacturer's instructions. Briefly, cells treated with or without siRNAs were lysed in 1× CHAPS lysis buffer; CHAPS lysis buffer is 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM EGTA, 0.1 mM benzamidine, 5 mM β-mercaptoethanol, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, and 10% glycerol. Cells were then extracted according to the manufacturer's instructions. The protein concentration of each cell lysate was measured by the Bradford method and adjusted with 1× CHAPS lysis buffer so that the concentration was equal across samples. The adjusted lysates were further diluted, and a diluted (1:20) extract was used to detect telomerase activity by telomeric repeat amplification protocol (TRAP) using a 32-P-labeled substrate primer. The amplified products from TRAP assays were resolved in an 8% native polyacrylamide gel electrophoresis (PAGE) gel, dried, and exposed to X-ray film. Telomerase activity was calculated as the percentage of the total intensity of the telomerase product ladders divided by the sum of the total intensity of the telomerase product ladders plus the remaining standard internal control intensity.
Various plasmids containing HPV16 or HPV18 E6 and E7 ORFs were cloned in frame into a mammalian expression vector, pFLAG-CMV-5.1 (Sigma), at the EcoRI and Asp718 sites of the polylinker region. The resulting plasmids produce a fusion protein with a FLAG tag on its C terminus. Plasmid pTMF54 containing HPV16 nt 104 to 855 was derived from plasmid pTMF1, which has a GU-to-GG mutation in the E6E7 nt 226 5′ ss (41). Plasmid pTMF58 is an HPV16 E6*I cDNA construct derived from CaSki cell RNA and contains HPV16 E6* I from nt 104 to 855 but is missing the E6 intron (nt 227 to 408). pTMF58 also has a FLAG tag introduced in frame at the N terminus of the spliced E6 ORF. Plasmid pST101 was derived from pTMF58 but has a deletion of 143 nt from the noncoding region between E6*I ORF and E7 ORF. Plasmid pWX1, which has a wild-type (wt) HPV16 insert at nt 81 to 880 in the N terminus of GFP in the pEGFP-N1 vector, was described in our previous publication (36, 41) and has no FLAG tag on either end. Plasmids pTMF56, pTMF59, and pZMZ84 all contain an 18E6E7 sequence derived from HeLa cells. Similar to pTMF54, plasmid pTMF56 contains HPV18 at nt 105 to 904 with a GU-to-GG mutation in the E6E7 nt 233 5′ ss. Plasmid pTMF59 is an HPV18 E6*I cDNA construct that contains HPV18 from nt 105 to 904 but has no E6 intron (nt 234 to 415). This plasmid also has a FLAG tag introduced in frame into the N terminus of the spliced E6 ORF. Plasmid pST102 was derived from pTMF59 but has a deletion of 122 nt from the noncoding region between E6*I ORF and E7 ORF. Plasmid pZMZ84 contains an HPV18 (nt 103 to 967) insert in the N terminus of GFP in pEGFP-N1 vector and has no FLAG tag on either end. For GFP reporter assays, pZMZ69 (HPV18) and pZMZ70 (HPV16) were used; these contain the E6 and E7 coding regions from each virus, respectively, fused in frame to the C terminus of GFP and were described previously (36).
293 cells were cotransfected with 2 μg of a GFP-HPV16 E6E7 fusion reporter (pZMZ70) or a GFP-HPV18 E6E7 fusion reporter (pZMZ69) plus various doses of individual siRNAs in the presence of Lipofactamine 2000 (Invitrogen). Twenty-four hours after transfection, the cells were collected for flow cytometry analysis.
293 cells (5 × 105 cells) transfected with 2 μg of HPV16 plasmid (pTMF54, pTMF58, pWX1, or pST101) or HPV18 plasmid (pTMF56, pTMF59, pZMZ84, or pST102) were examined for viral RNA splicing and E7 production. Total-cell RNA and protein were prepared 24 h after transfection. For protein sample preparation, 250 μl of 2× SDS loading buffer was used to directly lyse the cells, and 50 μl of the samples was used for a Western blot assay. Total RNA was prepared with TRIzol protocol (Invitrogen), and RNA splicing of HPV16 and HPV18 E6E7 pre-mRNAs in 1 μg of total-cell RNA was examined by RT-PCR using the same set of primers as described above for HPV16 (Pr106 plus Pr562 [see Fig. Fig.8]8] or plus Pr792 [oST196, 5′-A/ACGAATGTCTACGTGTG-3′; see Fig. Fig.9])9]) or HPV18 (Pr121 plus Pr850). E7 protein expressed from each plasmid was examined by Western blotting using anti-HPV16 E7 or anti-HPV18 E7. The same membrane was also probed for tubulin as a control for protein loading.
In HPV16 and HPV18, E6 and E7 are transcribed as a single bicistronic pre-mRNA that undergoes extensive alternative splicing. Our previous report (41) and results from several other laboratories (3, 9, 31) suggest that the E6E7 bicistronic pre-mRNAs are efficiently spliced in CaSki and W12-derived subclone cells (20861 and 20863), leaving the unspliced, full-length E6E7 mRNA at almost-undetectable levels. To further extend this observation, we examined CaSki (HPV16-positive), SiHa (HPV16-positive), and HeLa (HPV18-positive) cells for HPV16 and HPV18 E6 and E7 expression by an RPA in combination with an RT-PCR analysis. In this approach, two antisense E6E7 probes were designed to cover potential splicing junctions of the E6 coding regions in each E6E7 pre-mRNA (Fig. 1A and B). These probes were hybridized to total-cell RNA. As shown in Fig. Fig.1C,1C, the E6E7 pre-mRNAs were spliced efficiently in all three cell lines, producing mostly E6*I derived from splicing of the nt 226 5′ ss to the nt 409 3′ ss for 16E6E7 mRNAs or from splicing of the nt 233 5′ ss to the nt 416 3′ ss for 18E6E7 mRNAs. However, variations in splicing efficiency among cell lines became apparent by a quantitative analysis. Although both CaSki and HeLa cells produced abundant E6E7 transcripts, splicing of the E6E7 pre-mRNAs was more efficient in CaSki cells than in HeLa cells, with a higher ratio of E6*I/E6 in CaSki cells (7.4) than in HeLa cells (5.5). Compared to these two cell lines, SiHa cells transcribed at least seven times less E6E7 and had inefficient E6E7 RNA splicing, with an E6*I/E6 ratio of only 1.4. RT-PCR analysis also showed that the majority of the amplicons were E6*I mRNAs in SiHa and HeLa cells, although unspliced, intron 1-containing, full-length E6 mRNAs were also detected (Fig. 1D and E).
Using transient transfection, we previously demonstrated that splicing of the E6 intron from a bicistronic E6E7 pre-mRNA was important for the production of the E7 oncoprotein in 293 cells (41). To further confirm this observation in HPV-positive cervical cancer cells, we took an siRNA approach to specifically target a region that only exists in an unspliced, full-length E6 mRNA. We hypothesized that siRNA-mediated cytoplasmic degradation of the full-length E6 mRNA would not abolish expression of the E7 oncoprotein if E6*I was the mRNA used for E7 translation. Since the spliced E6*I mRNA does not have the intron 1 region, it should not be a target for this intron-specific siRNA.
HPV16-positive CaSki and HPV18-positive HeLa cells were chosen for the study, and two separate intron 1- and exon 2-specific siRNAs were created for each, as shown in Fig. 2A and B. Due to the bicistronic feature of the E6 mRNA, the targeted exon 2 regions that contain the E7 ORFs in the respective HPVs are also the 3′ untranslated regions (UTRs) of the E6 mRNAs. CaSki and HeLa cells were treated with effective low doses of intron-specific siRNA 209 for HPV16 (Fig. (Fig.2C)2C) and siRNA 219 for HPV18 (Fig. (Fig.2D).2D). These siRNAs did not affect E7 production but did specifically knock down E6, as signified by the stabilization of p53 expression. No high-quality anti-E6 antibody for HPV16 or HPV18 was available during the course of the study; instead, stabilization of cellular p53 protein was used as an indication of the siRNA-mediated suppression of E6 because high-risk HPV E6s induce p53 degradation. NS siRNA had no effect on E6 or on E7 in either cell type. The HPV16 intron 1-specific siRNA 209 had no effect on E7 expression in CaSki cells at 4 nM or 40 nM (Fig. (Fig.2C),2C), nor did the HPV18 intron 1-specific siRNA 219 have an effect in HeLa cells at 10 nM (Fig. (Fig.2D,2D, right). However, the HPV18 intron 1-specific siRNA 219 at a concentration of 40 nM did substantially suppress E7 (Fig. (Fig.2D,2D, left) due to its nuclear activity (see details below).
Treatment of HPV16-positive CaSki (Fig. (Fig.2C)2C) and HPV18-positive HeLa (Fig. (Fig.2D)2D) cells with the exon 2-specific siRNA 198 or siRNA 220 substantially suppressed E7 expression at both low (4 or 10 nM) and high (40 nM) concentrations. As the targeted exon 2 region is also the 3′ UTR of the unspliced, intron 1-containing E6 mRNA, both siRNA 198 and siRNA 220 also knocked down E6, as indicated by the stabilization of p53 expression, but they had less extensive effects on p53 stabilization than their corresponding intron 1-specific siRNAs. This is partially due to the presence of many more E6*I mRNAs than E6 mRNAs in the cells (Fig. (Fig.1C1C).
To confirm whether the observed alternation of E6 or E7 expression in siRNA-treated cervical cancer cell lines was related to the abundance of the unspliced, full-length 16E6 mRNAs, the same set of the HPV16 intron 1- or exon 2-specific siRNA was also applied to treat HPV16-positive SiHa cells which contain much more unspliced, full-length 16E6 (Fig. (Fig.1C).1C). As predicted, the suppression profiles of 16E6 or E7 by individual siRNA treatment of SiHa cells were very similar to those obtained from the HPV16-positive CaSki cells with similar treatment, despite SiHa cells expressing approximately 12 times less E7 (data not shown).
The difference in the effects of the HPV16- and HPV18-targeted siRNAs on E6 and E7 expression was not due to differences in their knockdown function. This conclusion was based on the effects of individual siRNAs on the GFP-16E6E7 or GFP-18E6E7 mRNA, which has inefficient RNA splicing due to an enlarged exon 1 (41). As shown in Fig. Fig.2E,2E, both siRNAs suppressed the expression of the associated GFP-E6E7 fusion, although the siRNAs for the GFP-HPV16 E6E7 fusion were more potent than the siRNAs for GFP-HPV18 E6E7 (compare Fig. Fig.2E,2E, top, to Fig. Fig.2E,2E, bottom). In each case, HPV-specific siRNAs were much better than a GFP siRNA in knockdown of the GFP fusion expression (Fig. (Fig.2E,2E, bottom, for HPV18; data not shown for HPV16), whereas an NS siRNA did not suppress expression at all and in fact appeared to stimulate the expression of the fusion proteins.
To double check the results that p53 is stabilized in CaSki and HeLa cells when an siRNA is used to suppress E6, we analyzed the expression of p21, a downstream target of p53, using multiple RNase protection assays with a set of cell cycle multiprobes. p21 transcription was significantly increased, presumably trans-activation by the accumulated p53, in each cell type treated with either an intron 1-specific or exon 2-specific siRNA (Fig. (Fig.3).3). Other cell cycle-related genes, including p53 and pRb, showed no change at the transcription level in these assays, demonstrating the specificity of p21 trans-activation by the accumulated p53 protein. Although both siRNA 209 (HPV16) and siRNA 219 (HPV18) were more potent in stabilizing p53 than siRNA 198 (HPV16) or siRNA 220 (HPV18) (Fig. 2C and D), the former two induced only slightly more p21 than did the latter two (Fig. (Fig.3,3, compare lanes 7, 19, and 24 to lanes 5, 18, and 23), suggesting that activation of p21 transcription does not require excessive p53 in these cells. It should also be noted that HeLa cells had lower levels of basal p21 transcription than CaSki cells (Fig. (Fig.3,3, compare lanes 9, 16, and 21 to lane 3) and therefore appeared to respond to p53 trans-activation of p21 more robustly, with an approximately five- to eightfold increase of p21 transcription in HeLa cells and only about a twofold increase in CaSki cells.
Given the fact that high-risk E7 interacts with hypophosphorylated pRb and induces its degradation (2, 4, 12), we then asked whether the siRNA-mediated inhibition of E7 expression could result in stabilization of pRb and its downstream targets. The status of p105Rb was examined by Western blotting in HeLa cells with or without siRNA treatment. As shown in Fig. 4A, a significant increase of hypophosphorylated p105Rb protein levels was observed with HeLa cells treated with 10 nM of exon 2-specific siRNA 220 but not in cells treated with an NS siRNA, indicating that suppression of E7 expression led to accumulation of the hypophosphorylated p105Rb. HeLa cells treated with the intron 1-specific siRNA 219 for 24 h also showed a moderate accumulation of hypophosphorylated p105Rb, due to the enhanced p21 expression (38).
The interaction of high-risk E7 with hypophosphorylated pRB leads to disruption of pRb-E2F complexes (6), releasing E2F from the complexes as a free transcription factor to activate expression of a group of genes, including cyclin A and E genes, that are essential for cell entry into S phase. Thus, expression of E7 triggers the activation of cyclin A expression (28), and suppression of E7 expression would decrease cyclin A expression. We therefore examined cyclin A levels in each cell line with or without various siRNA treatments. As expected, suppression of E7 expression by the exon 2-specific siRNA 220 in HeLa cells (Fig. (Fig.4B)4B) or siRNA 198 in CaSki (Fig. (Fig.4C)4C) reduced cyclin A expression somewhat. In contrast, interruption of E6 expression by the intron 1-specific siRNA 219 in HeLa cells or by siRNA 209 in CaSki cells, as with NS siRNA, had no effect on cyclin A expression (Fig. 4B and C).
Having demonstrated that reduction of E7 expression by exon 2-specific siRNA 198 or siRNA 220 stabilizes pRb and decreases cyclin A expression in cervical cancer cell lines, we hypothesized that more growth-suppressive pRb-E2F complexes would form in the cells with reduced E7 and consequently that blockade of the G1/S phase transition would occur. To test this hypothesis, we performed a cell cycle analysis by flow cytometry. Both intron 1- and exon 2-specific siRNAs promoted cell cycle arrest at G1 (Fig. 5A and B). Interruption of E7 expression from spliced E6*I mRNA produced the strongest cell cycle arrest at G1, with exon 2-specific siRNA 220 in HeLa cells (Fig. (Fig.5B)5B) having a stronger effect than siRNA 198 in CaSki cells (Fig. (Fig.5A).5A). In contrast, interruption of E6 expression from full-length, unspliced E6 mRNA by intron 1-specific siRNA 209 in CaSki cells (Fig. (Fig.5A)5A) and by siRNA 219 in HeLa cells (Fig. (Fig.5B)5B) produced only a moderate cell cycle arrest at G1 because of increased p21 expression due to p53 trans-activation. The percentage of sub-G0 events among the treatment groups ranged from 0.8% to 1.4% for CaSki cells and 1.2% to 1.3% for HeLa cells, indicating that the siRNA-induced cell cycle arrest did not relate to such minimal apoptosis in those samples. The observation of siRNA-induced cell cycle arrest was further supported by HeLa cell growth assays. Compared to nonspecific siRNA (NS siRNA), intron 1-specific siRNA 219 suppressed the growth of HeLa cells only moderately (32%), whereas exon 2-specific siRNA 220 conferred severe (63%) growth inhibition (Fig. (Fig.5C)5C) at the same concentration.
A moderate reduction of telomerase activity was observed in cell extracts of CaSki or HeLa cells that had been treated with either an intron 1- or exon 2-specific siRNA, compared to untreated cells. There was no difference in the reduction conferred by the intron 1-specific siRNA and the exon 2-specific siRNA in either type of cell (Fig. (Fig.6).6). These data are consistent with reports that repression of either E6 or E7 greatly reduces telomerase activity and c-Myc expression (7) and that E7 is required for E6-mediated telomerase activity (10).
To investigate whether siRNA-mediated suppression of E7 was due to siRNA-mediated degradation of E6*I RNA in HPV16-positive CaSki cells and HPV18-positive HeLa cells, we performed a quantitative RPA analysis using an antisense probe for each type of HPV. CaSki Cells treated with 40 nM of exon 2-specific siRNA 198 (Fig. (Fig.7A,7A, lane 4) had E6*I levels reduced by approximately 50%, an effect that was specific for siRNA 198 compared to intron 1-specific siRNA 209. At this dose, siRNA 209, which targets the intron 1 region, had only a minimal effect on E6*I mRNA (Fig. (Fig.7A,7A, lane 5). As expected, no reduction in levels of the intron 1-containing E6 was seen following treatment with siRNA 209 because so little such E6 was present in the cytoplasm and this E6 was indistinguishable from nuclear pre-mRNAs in our assay. Thus, it would be difficult to know even if there was some reduction on the intron 1-containing, cytoplasmic E6 mRNA by the siRNA 209. Together, these data fit well with the reduction of E7 expression by individual siRNAs seen with Western blotting (Fig. 2C and D), indicating that siRNA-mediated degradation of E6*I mRNA is responsible for the reduction of E7 expression.
Compared to CaSki cells, HeLa cells seemed more sensitive to HPV18-specific siRNA treatment. HeLa cells treated even with a low dose (4 nM) of exon 2-specific siRNA 220 (Fig. (Fig.7B,7B, lane 11) had E6*I levels reduced by approximately 70%. This specific effect on E6*I appeared more prominent at a relatively high dose (20 or 40 nM) (Fig. (Fig.7B,7B, lanes 4 and 9).
Although the exon 2-specific siRNA 220 in HeLa cells consistently reduced E6*I substantially (Fig. (Fig.7B),7B), as well as reduced the E7 protein levels (Fig. (Fig.2D),2D), the intron 1-specific siRNA 219 at a relatively high dose also unexpectedly reduced E6*I mRNA (Fig. (Fig.7B,7B, lanes 5 and 10) and E7 protein (at 40 nM) (Fig. (Fig.2D)2D) by 64% to 78%. Although its effect on E6*I was a little milder than that of the exon 2-specific siRNA 220 (Fig. (Fig.7B,7B, compare lanes 5 and 10 to lanes 4 and 9), siRNA 219-mediated reduction of E6*I mRNA in HeLa cells (Fig. (Fig.7B,7B, lanes 5 and 10) was dose dependent and cannot be explained by the current concept that siRNA functions in the cytoplasm. Since the E6*I mRNA is a spliced product that does not contain the target sequence of the intron 1-specific siRNA 219 and RNA splicing occurs only in the nucleus, reduction of the E6*I mRNAs by siRNA 219 must have occurred in the nucleus at the pre-mRNA level before RNA splicing. In addition, the intron 1-specific siRNA 219 showed no effect on various cellular mRNAs, including cyclophilin A (Fig. (Fig.7B)7B) and the many others shown in Fig. Fig.3,3, clearly eliminating the possibility that the reduction of the HPV18 E6*I mRNAs by the intron 1-specific siRNA 219 was due to an siRNA-mediated nonspecific RNA degradation, as has been previously reported (24, 27). To exclude the possibility that the reduction of E6*I mRNA by siRNA 219 was due to suppression of E6E7 pre-mRNA splicing or inhibition of viral RNA transcription in the nucleus, in vitro RNA splicing reactions were conducted with an 18E6E7 pre-mRNA containing intron 1 in the presence of various doses of synthetic, single-stranded, sense or antisense siRNA 219; nuclear run-on experiments were performed according to a published protocol (23) with cell nuclei prepared from individual siRNA-treated HeLa cells. The intron 1-specific siRNA 219 had no effect on RNA splicing (data not shown) or on RNA transcription initiation (data not shown). Thus, the reduction of the E6*I mRNAs in HeLa cells mediated by siRNA 219 must be a nuclear event in which the intron 1-specific siRNA 219 targets HPV18 E6E7 pre-mRNAs for nuclear degradation before RNA splicing.
Since the E6 stop codon is separated from the E7 start codon by only 2 nt in an intron 1-containing 16E6E7 mRNA and by only 8 nt in an intron 1-containing 18E6E7 mRNA, ribosomes might not be able to efficiently reinitiate E7 translation after termination of E6 translation from full-length, intron 1-containing E6E7 bicistronic mRNAs. Splicing of intron 1 from nt 226 to nt 409 in the 16E6 coding region or from nt 233 to nt 416 in the 18E6 coding region creates a frameshift and introduces a premature stop codon downstream of the splice junction. Therefore, the splicing increases distance (145 nt for HPV16 and 130 nt for HPV18) between translation termination codon of the E6*I and the E7 initiation codon AUG, which might benefit reinitiation of E7 translation. To test this hypothesis, six plasmids were constructed, three each from HPV16 and HPV18; their RNA transcripts are depicted in Fig. 8A and B. All six plasmids were then examined by transient transfection of 293 cells for their capability to translate E7. Notably, mRNAs derived from either plasmid pTMF54 for HPV16 or plasmid pTMF56 for HPV18, each of which contains a 5′ ss mutation, had no RNA splicing (Fig. (Fig.8C),8C), and produced almost no E7 (Fig. (Fig.8D).8D). In contrast, mRNAs transcribed from either plasmid pWX1 or plasmid pZMZ84, each of which contains a wt 5′ ss, underwent active RNA splicing (Fig. (Fig.8C)8C) and efficiently translated a remarkable amount of E7 from the spliced E6*I mRNAs (Fig. (Fig.8D).8D). This was further confirmed by using two different E6*I cDNA constructs derived from either CaSki (pTMF58) or HeLa (pTMF59) cells, which transcribed E6*I mRNA exclusively (Fig. (Fig.8C)8C) and produced a significant amount of E7 (Fig. (Fig.8D).8D). Considering these results, we concluded that the E6*I mRNAs are major RNA templates for production of the high-risk E7 oncoprotein.
To verify that the observed E7 production from spliced E6*I is truly a matter of distance between the upstream and the downstream ORFs in translation reinitiation, we constructed two separate expression vectors, pST101 and pST102, derived from 16E6*I cDNA (pTMF58) and 18E6*I cDNA (pTMF59), respectively, by deletion of the intercistronic noncoding region between the E6*I ORF and E7 ORF. The new constructs retain the same two ORFs, E6*I and E7, as do their parents, but utilize the E6 stop codon for their termination instead. Thus, the E6*I ORF in either pST101 or pST102 has the same distance as its corresponding E6 ORF from the E7 ORF (Fig. 9A and B). Transient transfection experiments show that keeping the E6*I ORF at the same distance as its E6 ORF from the downstream E7 ORF in pST101 or pST102 was detrimental for E7 production (Fig. (Fig.9C),9C), although both plasmids had active RNA transcription (Fig. (Fig.9D),9D), suggesting a translational deficiency of E7 from both the transcripts and the importance of the intercistronic distance between E6*I ORF and E7 ORF in the E6*I mRNAs in translation reinitiation of E7.
In this study, we used an siRNA approach to explore the complexity of the spliced 16E6E7 and 18E6E7 RNA isoforms and dissected their biological functions. We demonstrated that the majority of the viral oncoprotein E7 is derived from spliced E6*I mRNAs in cervical cancer cells, as well as in cells transfected with corresponding expression vectors. Full-length, bicistronic E6E7 mRNAs retaining intron 1 in the E6 coding region are responsible mainly for E6 production. Use of a synthetic, double-stranded, short RNA as siRNAs has been widely applied to the study of gene function (13). RNAi mediated by introduced synthetic siRNA duplexes has been used to silence HPV oncogene expression (5, 16, 40) with some success. However, lack of knowledge of bicistronic E6E7 RNA and its splicing in regulation of E6 and E7 translation make the results in those studies somewhat difficult to interpret. In this study, we designed all of our siRNAs based on our knowledge of the bicistronic 16E6E7 and 18E6E7 mRNA structures and alternative splicing. The siRNAs designed in this report have met at least six of eight criteria of siRNA design (25) and are highly effective and oncogene specific compared to other published siRNAs. Importantly, we were able to provide direct evidence of production of HPV16 or HPV18 E7 from spliced E6*I mRNAs. Use of RNA splicing to regulate E6 and E7 expression in high-risk HPVs might be a selective advantage to these viruses in carcinogenesis. Our findings on E6 and E7 production from a separate RNA transcript also provided us with a novel therapeutic intervention of individual oncogenes. It has been debated for decades how a high-risk E7 oncoprotein could be expressed from a bicistronic E6E7 mRNA. The main reason for this debate is that the E6 ORF in a bicistronic E6E7 mRNA is too close to the E7 ORF. Considering that ribosomes scan an mRNA without an internal ribosome entry site sequence in a linear fashion starting from the RNA 5′ end, this limited space between the E6 ORF and the E7 ORF would not give enough room or time for a scanning ribosome to release all of its termination components and to reload all of the necessary translation components to reinitiate translation of the E7 ORF on the same bicistronic mRNA (19). Because of this, two possible models have been proposed for how E7 could be translated: translation termination-reinitiation (29, 32) and leaky scanning-initiation (34). Our previous report and data from this study favor translation termination-reinitiation as the mechanism for high-risk E7 production based on the following evidence. (i) E6*I mRNAs expressed either from E6*I cDNA constructs or from an actively spliced E6*I mRNA produce abundant E7. (ii) Full-length, intron 1-containing E6 mRNAs that translate little or no E7 are present in minimal amounts in most HPV16- or HPV18-positive cervical cancer cells (3, 9, 31, 41). (iii) E6*I mRNAs are abundant transcripts in almost all HPV16- or HPV18-positive cervical cancer cells examined (3, 9, 31, 41). However, we do not exclude the possibility that a small fraction of minimal leaky scanning is also involved in translation of E7 from spliced E6*I mRNAs.
The translation termination-reinitiation model states that when the ribosome reaches the termination site of an upstream ORF, the 60S ribosomal subunit is released while the 40S subunit remains bound to the mRNA, resumes scanning, and initiates another round of translation at a downstream AUG codon. For the downstream reinitiation event to occur, the 40S subunit must reacquire Met-tRNAi; this is promoted by lengthening the intercistronic domain, which provides more time for Met-tRNAi to bind (18), or by increasing the concentration of eukaryotic initiation factor 2 (15). The efficient expression of E7 from E6*I cDNA constructs (pTMF58 for HPV16 and pTMF59 for HPV18) and the constructs that transcribe actively spliced E6E7 pre-mRNAs fits into this model well, since splicing of the E6 intron increases the distance from 2 (HPV16) or 8 (HPV18) nt to >130 nt between the two ORFs. This hypothesis was further supported by the data in this study that shortening the intercistronic distance of the E6*I ORF from the E7 ORF blocks E7 translation.
The leaky scanning model states that, when the first AUG codon occurs in a strong context, ANNaugN or GNNaugG, all or almost all ribosomes stop and initiate at that point. However, when the first AUG resides in a very weak context, lacking both R in position −3 of the AUG and G in position +4 of the AUG, some ribosomes initiate at that point but most continue scanning and initiate farther downstream. This leaky scanning enables the production of two separately initiated proteins from one mRNA (19). However, both the 16E6 and 18E6 AUGs are in a good context for ribosome recognition. The leaky scanning model proposed for E7 translation, which was mainly based on in vitro translation (34) and in vivo expression assays using a vaccinia virus vector that bypasses nuclear RNA splicing events (33), would therefore not explain why the plasmids pTMF54 (HPV16) and pTMF56 (HPV18), which transcribe the full-length E6E7 mRNAs with a 5′ ss mutation, expressed no detectable HPV16 and little HPV18 E7 (Fig. (Fig.8).8). This argument against the leaky scanning model on high-risk E7 production was further supported by our observation that making the E6*I ORF in the same distance as the E6 ORF upstream of the E7 ORF in an E6*I mRNA prevents E7 translation (Fig. (Fig.9).9). More obviously, the proposed leaky scanning model does not take account the E6*I mRNA as an RNA template for E7 translation, but instead argues for E7 production independent of RNA splicing (34).
The intron 1-specific siRNAs in this study induced not only extremely high levels of p53 and p21 but also a partial accumulation of hypophosphorylated p105Rb and cell cycle arrest at G1. Phosphorylation of the tumor suppressor protein pRb is dependent on Cdk, and p21 inhibits Cdk activity and interacts with E2F. Therefore, increased p21 expression in cells treated with the intron 1-specific siRNA would prevent hypophosphorylated pRb from being phosphorylated by Cdk (8, 14, 38, 39) and therefore from promoting cell cycle progression. However, high-risk E7 in HPV16- or HPV18-positive cells, in addition to interacting with hypophosphorylated pRb proteins, also forms a physical complex with p21 and inhibits p21 action (11, 17). Conceivably, the profound accumulation of hypophosphorylated p105Rb and substantial cell cycle arrest at G1 observed in the cells treated with the exon 2-specific siRNA was the outcome of all of those actions. It was noted (Fig. (Fig.22 and and3)3) that siRNA 198 and siRNA 220 can also induce p53 and p21, because their targeted regions are also the 3′ UTR of each E6 mRNA. The observed up-regulation of p53 and p21 expression by each exon 2-specific siRNA must therefore be a consequence of the degradation of the much less abundant E6 mRNAs, and a much higher percentage of the cells that were arrested at G1 phase in the siRNA 198-treated CaSki cells and in the siRNA 220-treated HeLa cells could be attributed to coexistence of more growth-suppressive pRb-E2F complexes and an enhanced expression of p21 in these cells.
The principle of how siRNA functions has been gradually elucidated and the pathway by which an siRNA mediates degradation of targeted mRNAs has been established. In general, siRNA-mediated RNA degradation occurs in an RNAi-induced silencing complex in the cytoplasmic mRNA decay center, P-bodies (20, 30). In this study, an intron 1-specific siRNA 219 was found, at a relatively high dose, to target HPV18 E6*I mRNAs for degradation in HeLa cells. Because E6*I lacks intron 1, the targeting must have occurred at the pre-mRNA level, before RNA splicing in the nucleus. Since the intron 1-specific siRNA 209 has only minimal activity in CaSki cells, this suggests that the observed nuclear activity varies from cell type to cell type, most likely due to differences in the nuclear membrane structures and other variables. While the manuscript was in preparation for publication, Robb et al. reported a similar observation of siRNA-mediated nuclear degradation of targeted nuclear RNAs (26). Together, these independent findings clearly indicate that a functional siRNA pathway exists in the nucleus, at least in HeLa cells.
This research was supported by the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, NIH.
We thank Catharine Smith at NCI for providing her nuclear run-on protocol and the β-actin plasmid and Douglas Lowy, Robert Yarchoan, Carl Baker, and Adrian Krainer for their comments, suggestions, and critical reading of our manuscript.