Fig. 7

U12 circular RNA has tissue-specific increases in development. In the gene diagrams of (a, d), annotated exons are shown as gold boxes, un-annotated “cryptic” exons as gray boxes. Definitive U12-type introns are indicated by “U12” in green; other introns are U2-type (or possibly cryptic U12-type). Splice-signal dinucleotides are shown in red. a Our de novo algorithm identified two circular isoforms in RANBP17 that use the U12-type splice signal following exon 20; these were validated by PCR and clone sequencing (which also identified the third isoform shown). b By RT-qPCR, the de novo RANBP17 circular isoforms show induction during fetal development in all tissues examined; the expression varies between tissues, for example, being significantly higher in the heart than the intestine. Values plotted are ΔΔCt = ΔCt(20 weeks) – ΔCt(10 weeks), where ΔCt = Ct(ACTB) – Ct(RANBP17); error bars are standard error of the mean of technical replicates. c The fraction of RANBP17 transcripts that are circular isoforms increases over developmental time. Shown are the percentages of each RANBP17 isoform, based on RNA-Seq junctional reads, at two different time points in fetal heart development. “circle1” = chr5:170632616:170610174, “circle2” = chr5:170632616 :170610198 (hg19 genome junctional coordinates; the third circle was not included in this analysis). Total junctional read counts were 240 and 267 for 19 and 28 week samples, respectively. d The de novo algorithm identified a circular junction in ATXN10, between the U12-type splice signal following exon 10 to a specific site within intron 9. PCR and clone sequencing with outward-facing primers in exon 10 verified the junction and also showed that additional un-annotated exonic sequences also form part of these circular isoforms, which show alternative splicing. Pathological expansion of a short repeat within intron 9 is a genetic hallmark of spinocerebellar ataxia type 10; the repeat region, marked with a red triangle, lies close to exonic sequences that we have identified as contributing to the circle