circRNAs enriched in nascent RNA fractions. (A) Nascent RNA (red) is collected from HUVECs following nuclei purification, and DNase I and Group-III caspase treatment to detach transcription factories (gold) from the substructure. (B) Heatmap showing circRNA enrichment over their linear counterparts (DCC-ratio) in factory-seq data. circRNAs with a ratio >0.5 are indicated (starred). (C) Factory and total RNA-seq coverage along CAMSAP1 and RAB3IP. Zoomed-in view: signal enrichment between circularized exons. (D) Bar graph showing RT-qPCR mean fold enrichment (from two replicates) of RNase R-treated over untreated samples. (*) P < 0.05; unpaired two-tailed Student's t-test. (E) Relative circRNAs and mRNA enrichment in polysome fractions normalized to whole-cell levels (±SD, from three replicates) determined by RT-qPCR. PL: light polysomes; PH: heavy polysomes. (F) Decay plots of the RNAs from panel E following transcriptional inhibition for 0–24 h. RNA levels (±SD, from three replicates) were normalized to YWHAZ and plotted relative to 0-h levels. Half-lives were CAMSAP1mRNA = 1.9 h, circCAMSAP1 = 32.2 h, RAB3IPmRNA = 3.8 h, circRAB3IP = 4.6 h. (G) Representative circCAMSAP1 and circRBA3IP FISH signal (arrowheads) in HUVEC counterstained with DAPI. The nonnascent RNA-enriched circHSGP2 signal provides a control. Bar: 10 µm. (H) Bar plots showing percent of cells (from two experiments) without focal circRNA FISH signal (gray), with at least one cytoplasmic (green), or at least one nuclear focus (blue) in images like those in panel G.
