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. 2019 Sep 6;20:237–247. doi: 10.1016/j.isci.2019.08.058

Figure 2.

Figure 2

Spliceosome Inhibition Leads to Expression Changes of circRNAs and Their Parent mRNAs

(A) Scatterplot showing the log2 fold change (FC) of 776 robustly expressed circRNA (y axis) and their parent mRNA (x axis) between Isoginkgetin and control conditions. Magenta dots indicate circRNAs that are significantly up-regulated in Isoginkgetin. The shade of magenta (from light to dark) indicates whether the parent mRNA was down-regulated, unchanged, or up-regulated, respectively. Green dots represent significantly down-regulated circRNAs, with the parent mRNA being also down-regulated (light green dots) or unchanged (dark green dots). Gray dots indicate circRNAs that are unchanged. Inset shows that the general abundance of circRNAs was higher than that of the parent mRNA after Isoginkgetin treatment (****p < 0.0001, unpaired t test, two-tailed).

(B) qRT-PCR validation of circHomer1, circGigyf2, circKlhl2, and circHook3 expression after Isogingketin treatment (**p < 0.01, unpaired t test, two-tailed, n = 3).

(C) qRT-PCR validation of parent Homer1, Gigyf2, Klhl2, and Hook3 mRNA expression (***p < 0.001, **p < 0.01, unpaired t test, two-tailed, n = 3).

(D) Validation of circRNA expression changes using high-resolution in situ hybridization for circHomer1 in control, Isoginkgetin, and Isoginkgetin and transcription-inhibitor treated neurons. Neuronal somata and dendrites were identified using anti-MAP2 immunostaining. Scale bar, 25 μm.

(E) Somatic circHomer1 expression was significantly up-regulated in Isoginkgetin-treated neurons compared with control. The inhibition of transcription prevented the Isoginkgetin-induced up-regulation of circHomer1 (***p < 0.0001, Kruskal-Wallis test followed by Dunn's multiple comparisons test, n = 73, 76, and 63).

(F and G) CircHomer1 expression after RNAi-mediated depletion of (F) SF3B1, (G) SF3A2, or scrambled control. The expression of SF3B1 or SF3A2 (magenta) in positively transfected cells (green) was validated after 4 days (***p < 0.0001, Mann-Whitney U test, n = 46, 39, 52, and 25). Scale bar, 25 μm.

(H) CircHomer1 fluorescence in situ hybridization after SF3B1 or SF3A2 depletion. Somatic circHomer1 was significantly up-regulated in SF3B1 and SF3A2 knockdown cells compared with scrambled control (***p < 0.0001, Mann-Whitney U test, n = 37, 63, and 57). Scale bar, 25 μm. Error bars represent SD. (B and C). Boxplot whiskers show minimum, first quartile, median, third quartile, and maximum (A, E, F, G, and H).