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

Identification and detection of HPV circRNAs

To screen for the presence of circRNA in HPV, we developed a pipeline to detect and visualize backsplice junctions from viral genomes (vircircRNA). To ensure accurate identification of all backsplices, circular viral genomes were concatenated and then utilized as the reference genome for the pipeline (Supplementary Fig. 1a–b). We selected ten HPV subtypes (Supplementary Fig. 1c) as reference genomes to screen against publicly deposited RNA-Seq datasets (Supplementary Fig. 1d). We identified 12 projects with RNA-Seq data from HPV-infected tissues. Despite the fact that most samples were not optimized for circular RNA sequencing through RNase R treatment or ribosomal RNA depletion, we identified 27 samples with multiple reads mapping to putative backsplice junctions (Supplementary Fig. 1e). In our initial screen, backsplice reads were identified from HPV16 and HPV35 (Fig. 1a, Supplementary Fig. 1e). Specific HPV16 circRNAs were notable because their abundance was comparable to spliced linear mRNAs (Fig. 1a, b). The vast majority (94%) of backsplice reads were generated from the head-to-tail joining of previously reported linear splice sites downstream in E1 (nt 16, E1^E4 splice donor) to one upstream in E6 (nt 7451, E6*I splice acceptor) (Fig. 1a–c, Supplementary Fig. 1e). This backsplicing is predicted to form a 472 nt circular RNA, which contains the entire open reading frame of E7 (Fig. 1c). Due to the established importance of the E7 oncogene and the abundance of HPV16 circE7, we focused on the characterization of this newly identified circRNA. We tested for the presence of backsplicing by inverse PCR using three cancer cell lines in which HPV16 has been shown to be stably integrated (CaSki and SiHa cervical cancer, UPCI-SCC154 tongue squamous cell cancer). RNase R is an exoribonuclease that specifically degrades linear, but not circular or lariat, RNAs. While a linear region of HPV16 E6/E7 was markedly decreased in abundance after RNase R treatment, the circE7 junction detected in all 3 cell lines before RNase R was enriched after treatment (Fig. 1d). Sanger sequencing of the amplified inverse PCR product from all three cell lines confirmed that the circE7 backsplice represented a true splice site rather than an intron lariat (Fig. 1e), which frequently contains untemplated nucleotides across the branch point¹³. HPV16 circE7 could be identified in the cancer cell lines by northern blotting as an RNase R-resistant band that migrated more slowly than its predicted size due to its circular structure¹⁴ (Fig. 1f). Quantitation of the northern blots revealed that circE7 represents ~1–3% of total E7 signals. Given the prevalence of HPV18 in HPV-induced cancers, we also tested 3 HPV18+ cell lines for the presence of a similar circular RNA (HPV18 circE7) (Supplementary Fig. 2a). While an analogous HPV18 circE7 could not be robustly detected by either RT-PCR or northern blotting (Supplementary Fig. 2b–c), RNA-Seq of SW-756 cervical carcinoma cells revealed several backsplice junction reads consistent with HPV18 circE7 as well as other potential circRNAs (Supplementary Fig. 2d).

Formation and translation of circE7

CircRNAs have been reported to function by sponging miRNAs^15–17. To determine whether circE7 might have a role as a miRNA sponge, we examined whether any miRNA-binding sites exist on the transcripts. While both HPV16 and HPV35 circE7s were predicted to have miRNA-binding sites (Supplementary Fig. 3a–b), none of the predicted miRNA-binding sites were conserved between the HPV16 and HPV35 circE7 species¹⁸. Since circRNAs have the potential to encode peptides, we next tested whether circE7 might be translated. To facilitate the detection of circE7 translation, we generated minigene expression vectors encompassing the entire ~1 kb backspliced region of the HPV16 genome (Fig. 2a). While the HPV sequence alone did promote circE7 generation, the addition of quaking (QKI) protein-binding sites, which have previously been reported to facilitate the circularization of RNAs¹⁹, enhanced E7 circularization by more than two-fold (Supplementary Fig. 3c). To determine whether circE7 had the potential to encode E7, constructs containing mutations of all three potential start codons (circE7_noATG) and E7 ORFs with a C-terminal 3xFLAG epitope tag were generated (Fig. 2a). Human embryonic kidney cells (HEK293T) were transfected with the minigenes and assayed for circRNA formation by RT-PCR using inverse primers. We detected RNase R-resistant circRNAs of the expected size from both WT and epitope-tagged circE7 (Fig. 2b). When HEK293T cells were transfected with the circE7 minigenes, we were able to detect E7 protein using both HPV16 E7-specific and FLAG antibodies, but not when the E7 start codons were mutated (Fig. 2c, d). The circE7 expression construct lacks the upstream E6 sequences required to generate any linear mRNAs previously reported to produce the E7 oncoprotein, including the E6*I isoform. To confirm that circular, rather than linear E7 RNAs, were responsible for E7 translation, we designed small interfering RNAs (siRNAs) to target sequences specific to the backsplice junction, the linear mRNA, or a region shared by both linear and circular species (Fig. 2a). RT-PCR confirmed that siRNAs against the circE7 backsplice preferentially knocked down the circular transcript; those against linear E6/E7 preferentially depleted the linear transcript; and those targeting E7 knocked down both RNAs (Supplementary Fig. 3e). Notably, knockdown of circE7 or shared regions of the E7 ORF inhibited the expression of E7 protein. In contrast, siRNAs targeting the linear RNA did not strongly decrease E7 expression by western blotting (Fig. 2c). CircRNAs are predicted to be translated through a cap-independent mechanism, which can be upregulated by cell stressors, including heat shock^20,21. 293T cells transfected with wild-type or FLAG-tagged circE7 constructs increased E7 translation, greater than four-fold and two-fold, respectively, in response to a 42 °C heat shock (Fig. 2d, Supplementary Fig. 3f). In contrast, a linear control RNA (FLAG-GFP) showed a greater than two-fold decrease in expression after heat shock (Fig. 2d, Supplementary Fig. 3f). To determine whether conserved splice sites were necessary for circE7 formation and expression, a circE7 minigene with mutations in both the splice acceptor and splice donor sites was generated (Fig. 2a–e; Supplementary Data 2). As expected, the splice site mutant constructs (circE7_SASD, circE7_FLAG_SASD) showed significantly lower levels of circE7 by both end point and RT-qPCR (Fig. 2e, f). E7 expression was markedly reduced after splice site mutation providing further evidence that circE7, rather than linear mRNAs, provided the template for E7 oncoprotein translation (Fig. 2g).

Molecular characterization of circE7

To determine the subcellular localization of circE7, cells were fractionated and nuclear and cytoplasmic fractions confirmed with MALAT1 and 18S or β-actin, respectively. Consistent with the behavior of other translated circRNAs^20,22–24, northern blots revealed that the majority of circE7 localized to the cytoplasm (Fig. 3a), and RT-qPCR confirmed the cytoplasmic enrichment of circE7 in both transfected 293 T cells (~58%) and CaSki cells (~75%) (Fig. 3b). Cap-independent translation of circRNAs has been reported to require N⁶-methyladenosine (m⁶A) modifications in the UTR²⁰. Because HPV16 circE7 possessed multiple potential m⁶A consensus sites (DRACH) (Fig. 3e), we performed m⁶A RNA immunoprecipitation (IP) experiments. Antibodies against m⁶A, but not an IgG control, pulled down circE7 with a comparable efficiency to SON, a control mRNA containing multiple m⁶A sites and previously confirmed to be methylated (Fig. 3c)²⁰. Consistent with the previously described role for METTL3/14 in circRNA methylation, siRNA-mediated knockdown of METTL3 resulted in decreased recovery of circE7 after m⁶A IP (Fig. 3d). Next, we constructed a circE7 mutant in which potential m⁶A motifs in the UTR were mutated (circE7_noDRACH) (Fig. 3e). Unexpectedly, this construct dramatically decreased the abundance of circE7, but not its linear E6/E7 counterpart (Fig. 3e, Supplementary Fig. 3g). Mutation of the potential m⁶A motifs strongly inhibited E7 oncoprotein expression, once again confirming the critical role for the circular, rather than the linear, RNA in E7 translation (Fig. 3f). Finally, we performed polyribosome (polysome) fractionation to determine whether circE7 might be associated with polysomes as has been reported for other translated circRNAs^20,23. Analysis of monosome (M), light polysome (L), and heavy polysome (H) fractions revealed circE7 to be associated with all fractions. Mutation of the start codons in circE7, circE7_noATG, abrogated the association of circE7 with heavy polysomes and attenuated its presence in the monosome and light polysome fractions. The association of a control mRNA (β-actin) was not affected (Fig. 3g). Thus, circE7 is m⁶A-modified, enriched in the cytoplasm, associated with polysomes, and capable of generating the E7 oncoprotein in a heat-shock regulated manner.

Functional characterization of circE7 in cancer

The functions of most circRNA remain ambiguous. In particular, the possible functions of virus-encoded circRNAs and those purported to code for proteins remain poorly characterized. To determine the biological functions of circE7, we depleted circE7 in CaSki cells using two doxycycline (Dox)-inducible short hairpin RNAs targeting the circE7 backsplice junction (circE7 sh1/2). After lentiviral transduction of the circE7 shRNA-expressing plasmid, we confirmed the specificity of the circE7 shRNA by RT-qPCR. After Dox induction, both circE7 shRNA resulted in a significant reduction of circE7 levels as assessed both by RT-PCR and northern blotting (Fig. 4a, b). Importantly, we did not note a significant reduction of the linear E6/E7 sequences or levels of the E6*I transcript (Supplementary Fig. 4a–c). Unexpectedly, both RT-qPCR and northern blots suggested that circE7 knockdown actually caused an increase in linear HPV16 E6/E7 transcripts (Supplementary Fig. 4a–b). Next, we tested whether loss of circE7 would impact levels of E7 protein in CaSki cells. Induction of circE7 shRNA 1/2 (sh1/2) decreased levels of endogenous E7 protein by greater than two-fold (Fig. 4c, Supplementary Fig. 4d), demonstrating that circE7 is required for optimal E7 expression in CaSki cells. CircE7 knockdown did not significantly decrease levels of the E6 oncoprotein (Fig. 4c, Supplementary Fig. 4e). Consistent with E7′s established role in transformation, depletion of circE7 resulted in decreased cell proliferation as measured by both cell number and MTT assay (Fig. 4d; Supplementary Fig. 4f-g). CaSki cells expressing circE7 shRNA showed significantly decreased entry into S phase as measured by BrdU incorporation (Fig. 4e, Supplementary Fig. 4h) consistent with a critical role for E7 in overriding Rb’s function in regulating cell cycle progression²⁵. Induction of circE7 sh1/2 also significantly inhibited the ability of CaSki cells to form colonies in soft agar (Fig. 4f). To confirm that sh1/2 did not impact CaSki proliferation through off-target effects, a circE7 resistant to shRNA (circResist_WT) was generated by including point mutations in the backsplice junction region while splice site consensus residues were not altered (Supplementary Fig. 5a). To determine whether the protein-coding capacity was required for the function of circE7, a shRNA resistant circE7 lacking start codons was also generated (circResist_noATG) and cloned. CaSki cells were doubly transduced with either vector control, circResist_WT, or circResist_noATG and also the Dox-inducible circE7 sh1/2 vectors (Supplementary Fig. 5a). As expected, while both circResist_WT and circResist_noATG rescued the expression of circE7 by RT-qPCR, only circResist_WT enhanced the expression of the E7 oncoprotein and rendered it resistant to circE7 sh1/2 knockdown (Supplementary Fig. 5c–f). Notably, expression of circResist_WT fully rescued CaSki growth after dox induction of circE7 sh1/2 (Fig. 4g). In contrast, circResist_noATG-expressing cells were able to rescue CaSki proliferation no better than the vector control (Fig. 4h, Supplementary Fig. 5b). In summary, the ability of circE7 to code for the E7 oncoprotein is absolutely essential for the transforming activity of circE7.

To further evaluate the biological functions of circE7, dox-inducible circE7 sh1/2- expressing CaSki cells were xenografted into immunodeficient mice for 21 days. The induction of circE7 sh1/2, through doxycycline in the drinking water, resulted in significantly smaller tumors (Fig. 5a, Supplementary Fig. 6a-b). Histological analysis of the harvested tumors showed that WT CaSki tumors showed tissue invasion, a high mitotic rate, and pleomorphic nuclei with a high nuclear-cytoplasmic ratio. In contrast, dox-induced CaSki tumors expressing circE7 shRNA were small, well-circumscribed, showed limited tissue invasion, and had a decreased nuclear-cytoplasmic ratio. Immunohistochemical (IHC) evaluation of tumors revealed that circE7 shRNA expressing tumors showed significantly less nuclear staining with the proliferative cell marker Ki-67 than the uninduced control (Supplementary Fig. 6c)²⁶. Finally, consistent with western blot analyses, IHC staining revealed markedly less nuclear and cytoplasmic staining for HPV16 E7 oncoprotein expression in CaSki cells after circE7 shRNA induction than the uninduced control. In summary, circE7 is essential for E7 expression and the transformed behavior of CaSki cervical carcinoma cells in vitro and in tumor xenografts.

Given the critical role of circE7 in maintaining the transformed phenotype of CaSki cells, we next determined whether circE7 might be more broadly relevant in human cancers. We analyzed TCGA RNA-Seq data from the mRNA pipeline for cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and head and neck squamous cell carcinoma (HNSC) using the vircircRNA pipeline. More than 100 patient tumors with at least two reads matching the same HPV backsplice junction were identified. To ensure the specificity of the identified HPV reads, a control analysis of kidney renal clear cell carcinoma (KIRC), which has been reported to be free of HPV transcripts²⁷, yielded no HPV sequence reads or backsplice junctions using identical search parameters. Backsplice junctions were detected from multiple high-risk HPVs, with HPV16 being the most abundant species (Fig. 5c, Supplementary Data 1). Consistent with our preliminary pipeline analysis, multiple species of circE7 were, by far, the most abundant type of backsplice identified in all high-risk HPVs ( >95% of total species). In contrast to the limited detection of circE7 from HPV18 in our in vitro analyses, HPV18+ patient tumors also showed frequent backsplices consistent with HPV18 circE7. As most of the TCGA RNA-Seq samples underwent polyA enrichment, the number of samples and abundance of the circRNA in the TCGA dataset are likely greatly underestimated.

To begin to address whether circRNA expression might have broader relevance to the HPV life cycle, we tested whether circE7 could be detected in HPV BP, a human cervical epithelial cell line that maintains low levels of episomal HPV16. HPV BP contained detectable circE7 (Fig. 5d). In addition, we used a well-established in vitro model of HPV31 infection in which primary human foreskin keratinocytes were transfected with a religated HPV31 genome²⁸. HPV31-transfected keratinocytes and an epithelial cell line derived from a grade II cervical intraepithelial neoplastic (CIN) lesion both had detectable circE7 (Fig. 5e). Notably, differentiation of these epithelial cells by high calcium resulted in significant decreases in the levels of circE7 in both HPV31-transfected cells and CIN612 cells²⁹. These experiments suggest that circE7 may play a role in the HPV life cycle and could be regulated by keratinocyte differentiation, similar to other transcripts from the HPV genome.

De novo circular RNA detection from circular viral genomes

To detect and display circular RNA from RNA sequencing (RNA-Seq) data of viruses with circular genomes, a custom pipeline named vircircRNA was developed (https://github.com/jiwoongbio/vircircRNA). Because the arbitrary linearization of circular genomes causes confusion in distinguishing between back and forward splices, two genomes were concatenated and used as the reference sequence for read mapping. RNA-Seq reads were aligned using Burrows-Wheeler Aligner (BWA, v0.7.15)³⁵ with specific options, “-T 19” to reduce minimum score to output and “-Y” to use soft clipping for supplementary alignments. Reads mapped to different positions with soft clipping were extracted as candidate segmented reads by splicing. Canonical GT-AG splice donor-acceptor motifs were used to exclude non-splice reads and define splice breakpoints. Information on strand specificity of sequencing and annotation of genes and promoters were included if available. Splice reads mapped in chiastic order were defined as back-splice reads from circular RNAs. The back-splice junction ratio was calculated by employing the equation:

where N_{back−splice}, N_{5′forward−splice} and N_{3′forward−splice} are the numbers of back-splice, 5′ and 3′ normal-splice junction reads.

Circular RNA detection from public and TCGA RNA-Seq data

For selected HPV, the genome sequences and the annotations were downloaded from National Center for Biotechnology Information (NCBI) nucleotide database. We used the keywords of “HPV” and “human papillomavirus” to search public sequencing data of HPV from NCBI Sequence Read Archive (SRA) and found 154 RNA-Seq data accessions of cDNA library from 12 projects. For the TCGA data, we downloaded raw sequencing data from cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC, 309 samples), head and neck squamous cell carcinoma (HNSC, 566 samples), and kidney renal clear cell carcinoma (KIRC, 618 samples) from The Cancer Genome Atlas (TCGA). To avoid false mapping, reads (50–51 bp) were mapped onto both HPV genomes and human transcript sequences downloaded from Ensembl (release 92, GRCh38) (https://www.ensembl.org/). Only reads with alignment scores on HPV genomes greater than those on human transcripts were kept for further analysis.

Cell culture

C-4I cells (gift from Diego Castrillon) were cultured in Waymouth’s and 10% FBS. CaSki cells (ATCC CRM-CRL-1550) were cultured in RPMI and 10% FBS. HeLa (ATCC CCL-2), SW756, and SiHa (gift from Diego Castrillon) cells were cultured in DMEM and 10% FBS. UPCI-SCC154 (ATCC CRL-3241) cells were cultured on Collagen I coated plates in DMEM/F-12 (Gibco, 11320082) supplemented with GlutaMAX (Gibco, 35050061), 10% FBS, 0.1 μg/ml hydrocortisone (Sigma, H0888), and 10 ng/ml EGF (Invitrogen, 10450–013).

Northern blots

Total RNA from indicated cells was extracted by Qiagen RNeasy. Similar results were obtained using TRIzol (Invitrogen). For cancer cell lines, 20–30 µg of total RNA was treated with RNase R or 8 µg of total RNA used for mock treatment. Similar results were obtained with using rRNA depleted samples (NEBNext) with 4ug of depleted RNA for each sample. For fractionated RNA sample, 4 µg of total RNA per fraction was used. RNA samples were heated in 2× formamide sample buffer and were electrophoresed in 1.5–2.0% formaldehyde-agarose gels in MOPS buffer and transferred onto Hybond N+ membranes (Amersham) with 10xSSC. Probe fragments were generated by PCR using the primers indicated in Supplementary Data 2. PCR products were gel purified (Machery Nagel) and used to generate α-³²P dCTP-labeled probes using the Random Prime Labeling Kit (Takara/Clontech). Probes were purified over nucleotide purification columns (Zymo Research) prior to hybridization. Hybridization was performed in PerfectHyb buffer (Sigma) at 65 °C overnight and washed according to Hybond’s instructions. Blots were developed on PhosphorImager screens and developed on a Typhoon Imager. Blots were quantited with ImageQuant.

Circular RNA minigene expression constructs

Circular E7 RNA cDNA were synthesized as gBlock fragments (IDT), PCR amplified, and cloned into pcDNA3.1- vector by NotI/BamHI restriction sites. Positive clones were verified by Sanger Sequencing (Genewiz). Constructs were transiently transfected into LentiX-293T (Clontech) cells by Lipofectamine 3000 including the P3000 reagent (ThermoFisher). At 48–72 h post-transfection, cells were harvested for fractionation, RNA preparation, or WB as indicated. For siRNA, 293T cells were plated onto 6-well plates 24 h before transfection to target a density of 30% at the time of transfection. Cells were co-transfected with 60 pmol of the indicated siRNA and 4 μg of the indicated vector without the P3000 reagent. Cells were harvested 68–72 h post-transfection for RNA preparation, WB, or m⁶A IP.

Western blotting

Whole-cell protein extracts were separated on SDS-PAGE gels, transferred to PVDF membranes and probed with the following primary antibodies: anti-FLAG HRP (1:300, Sigma, A8592), anti-GAPDH (1:1200, Santa-Cruz, sc-32233), anti-HPV16 E6 (1:200, GeneTex, GTX132686), and anti-HPV16 E7 (1:700, GeneTex, GTX133411). Membranes were then incubated with the appropriate HRP-conjugated secondary antibody (1:000, anti-mouse IgG, Cell Signaling Technologies, 7076P2; 1:1000, anti-rabbit IgG, ThermoFisher, G21234) and developed with an ECL system (Perkin Elmer, NEL104001). CaSki cells were harvested and lysed in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate) in presence of 1× protease/phosphatase inhibitor. Cells were incubated on ice with RIPA buffer for 10–15 min. Whole-cell lysate was then cleared by centrifugation at 12,000 × g for 15 min. The cleared supernatant was transferred to new tubes, before added Laemmli sampling buffer and boiled for at 95 °C for 10 min.

RNA isolation, RNase R treatment, cDNA synthesis

Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, 74104) and RNase R digested³⁶. Similar results were obtained with TRIzol reagent (Sigma, T9424). A concentration of 2 µg of total RNA was incubated with 5U RNase R (Lucigen, RNR07250), 10U murine ribonuclease inhibitor (New England Biolabs, M0314S), 0.5U DNase (Qiagen, 79254), and 1X RNase R buffer for 40 min at 37 °C and then placed on ice. Water was substituted for RNase R in mock reactions. 1 µl of 1 mM EDTA and 1 µl random hexamer (100 µM) were added to the unpurified RNase R digested products and denatured at 65 °C for 5 min. Samples were then placed on ice and reverse transcribed using the Superscript IV RT system (ThermoFisher, 18091050). For standard cDNA synthesis (no RNase R), 1 µg of total RNA was then reverse transcribed using Bio-Rad iScript cDNA synthesis kit according to manufacturer’s instruction.

End point PCR and RT-qPCR

End point PCR was performed with SapphireAmp (Takara, RR350B) by denaturation at 95 °C for 5 min, followed by 23 cycles at 95 °C 1 min, 62 °C 30 s, 72 °C 1 min, and a final extension step at 72 °C for 7 min for linear products. Cycling conditions for circRNA: were as follows: 95 °C 5 min, followed by 40 cycles of 95 °C 1 min, 62 °C 1 min, 72 °C 2 min, and a final elongation step at 72 °C for 10 min. Cycling conditions for linear mRNA: 95 °C 5 min, followed by 23 cycles of 62 °C 30 s, 72 °C 1 min, 72 °C 7 min, 23 cycles. For end point PCR, cDNA templates were diluted 1:4 in water for linear PCR reactions. For RT-qPCR, cDNA products were diluted 1:20, 2 µL of diluted DNA sample was used as template for real time PCR analysis with PowerUp SYBR Green (Applied Biosystems, A25779). Data were normalized by β-actin as a loading control and then presented as relative normalized expression level.

Nuclear and cytoplasmic fractionation

Cells grown in 35 mm dishes were trypsinized and centrifuged at 500 × g for 3 min at 4 °C. Pelleted cells were washed once with ice-cold PBS, centrifuged again, and resuspended in 250 µl of ice-cold Buffer I [0.5% Triton X-100, 0.32 M sucrose, 3 mM CaCl₂, 2 mM MgCl₂, 0.1 mM EDTA, 10 mM Tris (pH 8.0), 50 mM NaCl, 1 mM DTT, 0.04 U/µl RNase inhibitor (ThermoFisher, 18091050)]. After a 15 min incubation on ice, cells were centrifuged at 500 g for 5 min at 4 °C. The supernatant was collected for the cytoplasmic fraction and the pellet was resuspended in 250 µl of Buffer I for the nuclear fraction. RNA was extracted from the cytoplasmic and nuclear fractions using the RNeasy Mini Kit (Qiagen, 74104) and reverse transcribed with Superscript IV RT system (ThermoFisher, 18091050) using random hexamers.

m⁶A RNA immunoprecipitation assay

pCDNA3.1- empty vector or circE7 expression constructs were transiently transfected into LentiX-293T cells in triplicates. Total RNA was harvested 48–72 h post-transfection. 5 μg total RNA was immunoprecipitated (IP) by 1 μg m⁶A polyclonal antibody (Synaptic Systems#202003)³⁷. Immunoprecipitated RNA was then purified over QIAgen RNeasy columns. Purified immunoprecipitated RNA, along with 10% input RNA were then reverse transcribed by Bio-Rad iScript DNA synthesis kit and analyzed by real time PCR. Percentages of m⁶A modified RNA for both circE7 were calculated based on the input reading.

Polysome purification and RNA preparation

293T cells were plated in 10 cm plates 24 h before transfection to target a density of 40–50% at the time of transfection. Cells were transfected with 10 μg of vector control (pcDNA3.1-), circE7_WT, or circE7_noATG with Lipofectamine 3000 according to the manufacturer’s instructions. After 48 h, the cells were treated with 100 μg/mL cycloheximide in DMSO at 37 °C, and then harvested by trypsinization for polysome profiling. Cells (~5–6 × 10⁷) were lysed in 2 mL of polysome lysis buffer (20 mM Tris pH 7.4, 5 mM MgCl₂, 100 mM NaCl, 0.1% NP-40, 100 μg/mL cycloheximide) on ice for 15 min, in presence of EDTA-free protease inhibitor cocktail (ThermoFisher), followed by centrifugation at 4 °C for 10 min at 12,000 × g to pellet nuclei and mitochondria. The supernatant was then loaded onto a 10–50%(w/v) sucrose density gradient and ultracentrifuged at 20,000 × g for 2 h at 4 °C in a swinging bucket TH-641 rotor (ThermoFisher). The fractions were then collected by using BioLogic LP System (Bio-Rad). RNA was extracted from fractions using TriZol LS solution according to manufacturer’s instructions. For end point PCR analysis, 100 ng of RNA from each fraction was used for a standard RT-PCR reaction in 20 μL (ThermoFisher), and then 3 μL of undiluted reaction product was used for end point PCR.

Lentiviral, doxycycline-inducible shRNA circE7 constructs

Short hairpin RNA (shRNA) sequences specifically targeting the circE7 backsplice were designed using the RNAi Designer Program (https://rnaidesigner.thermofisher.com/). Forward and reverse oligos were annealed in NEB buffer 2, and then phosphorylated and ligated into EZ-Tet-pLKO-Blasticidin vector³⁸ (Addgene #85973) using NheI/EcoRI. For shRNA resistant rescue experiments (circResist_WT and circResist_noATG), shRNA resistant constructs were designed and synthesized by Integrated DNA Technology (IDT). Six bases were mutated within the shared shRNA target sequence. All mutations were transversions that were spaced evenly throughout the shRNA targeting sequence thus preventing any residual targeting by non-canonical basepairing or short complementary sequences, respectively. The shRNA resistant constructs were then cloned into a modified pLenti6.3-V5/DEST lentiviral vector. The backbone vector was modified to remove an attR recombination sequence as well as the V5 tag by SpeI + MluI digestion. The linearized vector was then re-ligated by a short oligo containing BamHI restriction site. The constructs were cloned into this modified vector through InFusion cloning with BamHI single digestion. To package lentiviruses, LentiX-293T cells were plated at ~70–80% confluence 12–16 h before transfection. Transfer plasmid, pMD2.G and psPAX2 were then co-transfected into LentiX-293T cells by Lipofectamine 3000 at molar ratio of 1:1:1. Viruses were harvested at 48 and 72 h post-transfection. For lentiviral transduction, CaSki cells were plated into 6-well plate at 50% confluence without any antibiotics. Viral supernatant was then added to the cell in presence of polybrene at final concentration of 8 μg/mL. For single infections (shRNA alone), the cells were then exposed to blasticidin selection 48 h after the intial transduction at a concentration of 15 μg/mL. Cells were selected for ≥7 days before the antibiotic concentration was decreased to 5 μg/mL for maintenance of stable cell lines. For double transductions (shRNA + shRNA resistant circE7), unmodified CaSki cells were lentivirally transduced simultaneously with the Dox-inducible shRNA constructs with a hygromycin selection marker and the shRNA-resistant constructs (circResist) with a blasticidin selection marker. The cells were then selected for more than ≥7 days with 100 μg/mL hygromycin and 15 μg/mL blasticidin. For shRNA expression induction, doxycycline was added to culture medium at final concentration of 1 μg/mL for at least three consecutive days and cells were then subjected to analyses.

Cell proliferation and MTT cell growth assay

Sixty-thousand (60,000) CaSki cells were plated on Day 0 in quadruplicate RPMI with 10%FBS (doxycycline free) with or without 1 μg/mL doxycycline²⁶. Cells were counted daily after Day 2. For MTT-based cell growth rate analysis, 1000 CaSki cells stably transduced with two shRNA constructs were plated into each well of 96 well plates in triplicates for at least 8 days without doxycycline. The first time point was done 24 h after plating, serving as a reference point. After 24 h, doxycycline was added to the rest of plates. The MTT assay was performed daily for 7 days. All the readings from the same well were normalized by the reference point. MTT (Invitrogen#M6494) reactions were performed as recommended by manufacturer.

BrdU incorporation assay

CaSki cells stably transduced with circE7 shRNA1/2 were induced by 1 μg/mL doxycycline for at least three days. Control cells (no dox) and induced cells were then plated into Nunc 4-well chamber slides (ThermoFisher 154453) in triplicate. Cells were labeled with 10 μM BrdU for 1.5 h. Cells were then fixed by 4% paraformaldehyde at RT for 10 min, followed by 0.1% Triton X-100 permeabilization and 1.5 N HCl DNA hydrolysis. Cells were then probed by O/N incubation with BrdU antibody (1:400, BD BioScience) and stained with Alexa 488-conjugated secondary antibody (ThermoFisher). Samples were then subjected to DAPI nuclear counterstain (Vector Labs). BrdU positive cells were then quantified and data were presented as percentage BrdU positive cells.

Soft agar colony formation assay

CaSki cells stably transduced with the two doxycycline inducible shRNA constructs were induced by 1 μg/mL doxycycline for 3 days. 0.5% agar, DMEM, 10%FBS was plated as base layer in 6-well plate (35 mm). A total of 10,000 cells of each group were plated in 0.3% agar-DMEM, 10% FBS on top of the base layer in triplicates. Cells were fed with 500 μL RPMI with 10%FBS medium twice a week. Doxycycline (1 μg/mL) was included in all media for induced cells. Cells were then allowed to grow for 28 days before quantification. Colonies larger than 100 μm were scored.

CaSki xenograft assay

Xenograft assays were performed on eight week-old NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) (Jackson Laboratory 005557)³⁹. Mice were anesthetized and shaved on both flanks and injected subcutaneously with 4 × 10⁶ tumor cells in 200 μl of PBS. Four mice (n = 4) were used for each of the four group (sh1/2, −/+ doxycycline). For the Dox-induction group, the cells were pre-treated with 1 μg/mL doxycycline to prime shRNA expression one day before harvesting for injection. Dox-induction animals were given 1 mg/mL doxycycline water, which was changed every 2 days for the duration of the experiment. After 21 days, tumors were dissected and weighed. Tumors were fixed in 4% paraformaldehyde overnight at 4 °C and then moved to 70% ethanol. Immunohistochemical staining of paraffin-embedded tumor tissues was performed using Ki-67 (1:100) and E7 (1:100) primary antibodies. All animal procedures were performed in accordance with institutional guidelines and was approved by the Institutional Animal Care and Use Committee, animal protocol number 2015–101166.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.