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Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus

Zhao, Jiawei, Lee, Eunice E., Kim, Jiwoong, Yang, Rong, Chamseddin, Bahir, Ni, Chunyang, Gusho, Elona, Xie, Yang, Chiang, Cheng-Ming, Buszczak, Michael, Zhan, Xiaowei, Laimins, Laimonis, Wang, Richard C.·2019·Nature Communications
DOI 10.1038/s41467-019-10246-5PMC PMC6534539
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Abstract

Single-stranded circular RNAs (circRNAs), generated through ‘backsplicing’, occur more extensively than initially anticipated. The possible functions of the vast majority of circRNAs remain unknown. Virus-derived circRNAs have recently been described in gamma-herpesviruses. We report that oncogenic human papillomaviruses (HPVs) generate circRNAs, some of which encompass the E7 oncogene (circE7). HPV16 circE7 is detectable by both inverse RT-PCR and northern blotting of HPV16-transformed cells. CircE7 is N 6-methyladenosine (m 6 A) modified, preferentially localized to the cytoplasm, associated with polysomes, and translated to produce E7 oncoprotein. Specific disruption of circE7 in CaSki cervical carcinoma cells reduces E7 protein levels and inhibits cancer cell growth both in vitro and in tumor xenografts. CircE7 is present in TCGA RNA-Seq data from HPV-positive cancers and in cell lines with only episomal HPVs. These results provide evidence that virus-derived, protein-encoding circular RNAs are biologically functional and linked to the transforming properties of some HPV.

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Fig. 1 Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log2(read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (AE, red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northerns
Fig. 1 Identification of HPV circRNAs. a A transcript map generated by vircircRNA summarizing the splicing events identified for HPV16 from the combined SRA datasets (Supplementary Fig. 1e). Lines (top) indicate forward splicing events; arcs (bottom) indicate backsplicing; thickness = log2(read count); red arc highlights circE7. The lower panel represents a partial HPV16 genome with promoters (P, green arrowheads) and the early polyadenylation (AE, red line) indicated. Numbering from the NC_001526 reference sequence. b Alignment of sequencing reads spanning the circE7 backsplice junction from SRS2410540. Red indicates E7-E1 sequences, and blue indicates E6 sequence. c Predicted formation and size of HPV16 circE7. Arrows indicate primers used to detect linear E6/E7 and circE7. d RT-PCR of random hexamer primed total RNA from HPV16+ cancer cell lines. 2 μg of total RNA were treated with 5U of RNase R (or water for mock) in the presence of RNase inhibitor for 40 min prior to RT reaction. Results are representative of 4 independent experiments. e Sanger sequencing of PCR products from d confirmed the presence of the expected circE7 backsplice junction without the insertion of additional nucleotides. Sequencing traces were identical for 3 independent reactions from each cell line. f Northern blot of total RNA after mock (8 μg) or with RNase R treatment (20 μg) from the indicated HPV16+ cell line probed with HPV16 E7. Arrows indicates RNase resistant band with E7 sequence. Ethidium Bromide staining (bottom), RNase R treatment control. Results representative of 5 independent northernsCC BY · PubMed Central
Fig. 2 Generation and translation of circE7. a Diagram of constructs generated to express circE7 in vitro. Map indicates location of QKI sites, start codons (all mutated in ‘noATG’), 3xFLAG (present in ‘FLAG’), splice sites (mutated in ‘SASD’), and siRNA used in subsequent experiments. b RT-PCR confirms the formation of RNase R-resistant circE7 RNAs from transfected 293 T cells. Results representative of >6 independent experiments. c Western blot for FLAG from 293 T cells co-transfected with circE7_FLAG and the indicated siRNA. GAPDH, loading control. Results representative of 4 independent transfections. d Western blots for FLAG and HPV16 E7 from 293 T cells transfected with the indicated circE7_FLAG construct (4 μg) or a linear FLAG-GFP control vector (0.4 μg). Indicated transfections were subjected to a heat shock (2 h at 42 °C, 2 h recovery). Eight-fold less lysate was loaded for control FLAG-GFP transfected cells. GAPDH, loading control. Results representative of 5 independent experiments. e RT-PCR demonstrates that splice sites indicated in a are required for the formation of circE7 RNAs. f RT-qPCR from 293 T cells transfected with pcDNA3.1-circE7 constructs confirms significantly lower circRNA formation in the SASD mutants. Results representative of 3 independent experiments. g Western blots for HPV16 E7 from 293 T cells transfected with the indicated circE7 construct (4 μg). GAPDH, loading control. Source data for b, d, e, and g provided in Source Data file
Fig. 2 Generation and translation of circE7. a Diagram of constructs generated to express circE7 in vitro. Map indicates location of QKI sites, start codons (all mutated in ‘noATG’), 3xFLAG (present in ‘FLAG’), splice sites (mutated in ‘SASD’), and siRNA used in subsequent experiments. b RT-PCR confirms the formation of RNase R-resistant circE7 RNAs from transfected 293 T cells. Results representative of >6 independent experiments. c Western blot for FLAG from 293 T cells co-transfected with circE7_FLAG and the indicated siRNA. GAPDH, loading control. Results representative of 4 independent transfections. d Western blots for FLAG and HPV16 E7 from 293 T cells transfected with the indicated circE7_FLAG construct (4 μg) or a linear FLAG-GFP control vector (0.4 μg). Indicated transfections were subjected to a heat shock (2 h at 42 °C, 2 h recovery). Eight-fold less lysate was loaded for control FLAG-GFP transfected cells. GAPDH, loading control. Results representative of 5 independent experiments. e RT-PCR demonstrates that splice sites indicated in a are required for the formation of circE7 RNAs. f RT-qPCR from 293 T cells transfected with pcDNA3.1-circE7 constructs confirms significantly lower circRNA formation in the SASD mutants. Results representative of 3 independent experiments. g Western blots for HPV16 E7 from 293 T cells transfected with the indicated circE7 construct (4 μg). GAPDH, loading control. Source data for b, d, e, and g provided in Source Data fileCC BY · PubMed Central
Fig. 3 Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m6A or IgG control) after transfection with the indicated plasmid (24 h) (n = 8 biological replicates from 4 transfections). SON, m6A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m6A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. (n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data file
Fig. 3 Characterization of circE7. a CircE7-transfected cells were fractionated and indicated fractions analyzed by northern blot. Total RNA (4 μg) with mock or RNase R treatment of fractions from 293 T cells confirms that circE7 is enriched in the cytoplasm and is RNase R-resistant. MALAT1 and β-actin, fractionation controls. Band density (bottom) quantitated after normalization to the enriched fraction. Results are representative of 3 independent blots. b CircE7-transfected 293T (left) or untransfected CaSki (right) were fractionated and analyzed by RT-qPCR. MALAT1 and 18 S (top), fractionation controls. Values normalized to the enriched fraction. Results are representative of 3 independent fractionation experiments. c RT-qPCR of RNA IP (m6A or IgG control) after transfection with the indicated plasmid (24 h) (n = 8 biological replicates from 4 transfections). SON, m6A RNA IP control. d Western blot for METTL3 from 293T co-transfected with control or METTL3 siRNA and circE7 construct (top). GAPDH, loading control. RT-qPCR of RNA IP (m6A or IgG control) from 293 T cells co-transfected with indicated siRNA and circE7 construct. RT-PCR is representative of 4 independent experiments. e Schematic of the DRACH consensus motifs for METTL3/14 and the sites mutated in the circE7_noDRACH construct (top). RT-qPCR for circE7 in cells transfected with the indicated construct. Loss of UTR DRACH motifs in circE7 results in a significant decrease in the abundance of circE7, but not linear E6/E7. (n = 4 independent experiments). f Western blot for E7 from 293 T transfected with indicated circE7 construct. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with one-way analysis of variance (ANOVA) with Holm–Sidak tests. g Representative tracing of circE7-transfected cells after polysome enrichment assay with the monosome (M), light polysome (L), and heavy polysome (H) fractions indicated (left). Dashed lines indicate collected fraction. Detection of circE7 in polysome fraction by RT-PCR after transfection with circE7 or circE7_noATG (right). β-actin, control. Source data for a provided in Source Data fileCC BY · PubMed Central
Fig. 4 Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. (n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 104 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 104 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test (d, g, h) and one-way analysis of variance (ANOVA) with Holm–Sidak tests (a, e, f). Source data for b, c provided in Source Data file
Fig. 4 Protein encoding circE7 is essential for CaSki cell growth. a CaSki cells were lentivirally transduced with doxycycline (dox)-inducible hairpins specific for the circE7 backsplice junction (circE7 sh1/2). RT-qPCR for levels of circE7 revealed that circE7 sh1/2 resulted in significant decreases of circE7 levels. (n = 3 independent experiments, run in duplicate). b Northern blot of RNase R treated total RNA (30 μg) from CaSki cells with or without circE7 sh1/2 induction (2 days). Band density (bottom number) was quantitated and normalized to the uninduced control. c Western blots for E7 and E6 after circE7 sh1/2 induction (3 days). Western blots representative of 3 independent experiments. GAPDH, loading control. d A total of 6.0 × 104 CaSki cells were seeded in triplicate in six-well plates at day 0 and absolute cell number quantitated daily after day 2. CircE7 sh1/2 induction resulted in significantly slower growth of CaSki cells after day 4. Similar results were obtained in 3 independent experiments. e CaSki cells with or without circE7 sh1/2 induction (1 day) were plated in chamber slides and labeled with BrdU (10 μM for 1.5 h). Cells were stained with αBrdU and DAPI and scored as % of DAPI + cells. f 1.0 × 104 CaSki circE7 sh1/2 cells with or without induction (1 day) were seeded in triplicate in soft agar with or without dox (14 days). Average colonies per 35 mm. n = 4 independent transfections. g CaSki were doubly transduced with a shRNA resistant WT circE7 (circResist_WT) and circE7 sh1/2. MTT assay of circResist_WT cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. h CaSki were doubly transduced with a shRNA resistant circE7 with no start codons (circResist_noATG) and circE7 sh1/2. MTT assay of circResist_noATG cells with and without Dox induction. MTT values normalized to the uninduced (-Dox) condition. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test (d, g, h) and one-way analysis of variance (ANOVA) with Holm–Sidak tests (a, e, f). Source data for b, c provided in Source Data fileCC BY · PubMed Central
Fig. 5 Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×106), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice (n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test (a, e)
Fig. 5 Biological functions of circE7 in tumors and episomal HPV. CaSki cells (4×106), which had been stably transduced with the indicated construct, were xenografted onto the flanks of NSG mice (n = 8 per construct). Mice were given water with or without doxycycline (1 mg/mL) as indicated. a Image of representative CaSki tumor xenografts dissected from the indicated mice after 21 days (top). Weights of CaSki tumors with or without dox-induced circE7 sh1/2 expression (bottom). b Representative images of tumors formed by CaSki xenografts without (top) or with (bottom) doxycycline. Arrowhead indicates an area of invasive tumor. Arrows indicate mitotic figures and Ki-67-positive cells. Dashed box indicates area of detail. Scale bars, 200 μm. c TCGA RNA-Seq data (CESC, HNSC) was analyzed with vircircRNA and backsplices with ≥2 reads were tabulated. d RT-PCR from CaSki or HPV BP cells that possess integrated or episomal HPV16 genomes with or without RNase R reveals the presence of circE7 in both samples. e Human foreskin keratinocyte (HFK), keratinocytes infected with religated HPV31 (HFK + HPV31), or a HPV31 infected cell line derived from a grade II cervical biopsy (CIN612) were induced to differentiate with high calcium. Levels of HPV31 circE7 were assessed by RT-PCR (left) or RT-qPCR (right). Calcium-induced differentiation significantly decreased levels of HPV31 circE7. RT-PCR is representative of 4 independent experiments. Data are shown as mean ± s.d. P values (indicated above relevant comparisons) were calculated with two-tailed t test (a, e)CC BY · PubMed Central

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Metadata

Authors
Zhao, J., Lee, E. E., Kim, J., Yang, R., Chamseddin, B., Ni, C., Gusho, E., Xie, Y., Chiang, C.-M., Buszczak, M., Zhan, X., Laimins, L., & Wang, R. C.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-05-24
Volume
10
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-019-10246-5
PMC
PMC6534539
Cited by
276
References
5
Language
en
Update notices
correction (2022-05-18)
Licence
Funders
  • U.S. Department of Health & Human Services | National Institutes of Health (R01GM115473, R01CA103867, R01GM125812, R01CA059655, R01CA142861)
  • Welch Foundation (I-1805)
  • U.S. Department of Health & Human Services | NIH | National Cancer Institute (P30CA142543, P50CA70907)
  • Burroughs Wellcome Fund (1010978)
  • American Cancer Society (RSG-18-058-01)
  • U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR072655)

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Zhao, J., Lee, E. E., Kim, J., Yang, R., Chamseddin, B., Ni, C., Gusho, E., Xie, Y., Chiang, C.-M., Buszczak, M., Zhan, X., Laimins, L., & Wang, R. C. (2019). Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus. Nature Communications. https://doi.org/10.1038/s41467-019-10246-5