Fig. 5

Application of BEs for functional circRNA screening. A Computational screening of circRNAs with predominantly back-spliced exons could be targeted by hA3A-eBE-Y130F at back-splice sites. Thirteen circRNAs identified in at least two of three (ribo−, polyA−, or RNaseR-treated) RNA-seq datasets from 293FT cells were used for functional circRNA screening. B Cell viability revealed by MTT assay. Top: Schematic for cell proliferation assay to detect the effect of circRNAs on the cell growth. See “Methods” for details. Bottom: OD₄₉₀ values measured at day 0, 1, 2, 3, and 4 are listed. C Base changes at 3′ bss of the novel exon in the ZNF292 gene locus by hA3A-eBE-Y130F repressed the back-splice for circZNF292-nov. Top, Schematic of partial ZNF292 gene organization. Back-spliced novel exon was highlighted by pink bar. Context sequences of targeted 3′ (b)ss were shown by a, t, c, and g for intron or by A, T, C, and G for exon; Bottom, G-to-A base change ratio at targeted 3′ (b)ss of predominantly back-spliced novel exon in the ZNF292 gene locus. D Repression of circZNF292-nov by BE promotes 293FT cell proliferation, as revealed by MTT cell proliferation assays. Left, 3′ bss mutation decreases expression of cirZNF292-nov, but not expression of cirZNF292 or linear ZNF292 RNA(s). Right, cell proliferation ability revealed by MTT assays. E Repression of circZNF292-nov by BE promotes HCT116 cell proliferation. Refer to D for details. F circZNF292-nov knockdown by shRNAs also promotes 293FT cell proliferation, as revealed by MTT cell proliferation assays. D–F Error bar represents SD from three independent replicates. ns, not significant; ∗∗∗, P < 0.001; Student’s t test