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 (A_E, 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