Unannotated circRNA-predominant alternative cassette exons. (A) Hundreds of previously unannotated circRNA-predominant cassette exons (red) were identified in circRNAs from individual cell lines. (B) Previously unannotated circRNA-predominant cassette exons (red) were much less conserved than the annotated circRNA-predominant cassette exons (blue) or cassette exons in linear RNAs (gray). (C) Validation of circRNA-predominant cassette exons in PA1 cells by RT-PCR. Similar to the RNA-seq results (PSI ratio), semiquantitative RT-PCR showed the detection of six circRNA-predominant cassette exons from the p(A)− and p(A)−/RNase R RNA population but barely any from p(A)+ RNAs. The inclusion ratio of circRNA-predominant cassette exons from RT-PCR was determined by Quantity One (Bio-Rad). Note that circRNA-predominant cassette exons in PIP5K1C, PRRC2B, and RBPMS loci (bottom) were not previously annotated by RefGene, UCSC Known Genes, or Ensembl. Magenta bars, circRNA-predominant cassette exons. Blue bars, known exons. Divergent PCR primers were indicated as black arrows. (D) Validation of circRNA-predominant cassette exon inclusion from different cell lines by Northern blot on denaturing PAGE gels. The circRNAs with alternative cassette exon inclusion/exclusion in both PIP5K1C and PRRC2B loci were detected in PA1 and H9 cell lines. Note that the circRNA-predominant cassette exons in PIP5K1C or PRRC2B loci are previously unannotated. Magenta in the circles, circRNA-predominant cassette exons. Blue in the circles, known exons. (*) Linear RNA background. (E) Visualization of circRNA-predominant cassette exon inclusion in PIP5K1C locus from different cell lines. The inclusion ratio of the circRNA-predominant cassette exons from RNA-seq was indicated by PSI. Note that the circRNA-predominant cassette exon in the PIP5K1C locus is a newly identified exon in this study. Magenta bar, circRNA-predominant cassette exon. Blue bars, other exons.
