Shortening the Flanking MIRs Reduces circRNA Biosynthesis
(A) Schematic structures of MIR-deleted ciRS-7-expression plasmids. The numbers indicate the lengths of the deleted 5′ MIR elements.
(B) Generation of ciRS-7 from reporters, with various MIR lengths, in transiently transfected ciRS-7 knockout HEK293 cells. Total RNA extracted from DOX-treated cells was analyzed by Northern blotting. 28S rRNA was detected by ethidium bromide staining as a control. The band corresponding to ciRS-7 is indicated on the left (see Figure 2B legend for the upper products).
(C) ciRS-7 formation was also quantified by RT-qPCR. Expression levels were normalized to the level of precursor RNA (ciRS-7/Precursor) and plotted as ratios to the value of control wild-type (WT) plasmid-expressing cells. Therefore, these quantified results do not exactly match up to the apparent view of Northern data in (B). Means ± SD are given for three independent experiments (∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05).