Fig. 5: A-to-I editing enhances PTBP1 binding to intron and promote circCHEK2 biogenesis.
From: ADARs act as potent regulators of circular transcriptome in cancer
a In silico prediction of PTBP1 (red line) and TDP43 (black line) binding to unedited (WT) or triple mutated (edt123) RCM sequence of circCHEK2 using RBPmap. b WB analysis of RNA pull-down products showing the binding affinity of PTBP1 and TDP43 to the WT or edt123 RNA probes. c RIP-qPCR analysis of the binding of PTBP1 protein to the circCHEK2 RCM region in EC109 cells transfected with ADAR1 or empty vector control (EV). WB and qPCR analyses of PTBP1 RIP immunoprecipitates are shown in the left and right panels, respectively. d Pie charts illustrating the editing frequency (indicated by red slice) of each editing site (#1, #2, #3) in the indicated Input or RIP samples. Editing frequency of each editing site was measured using TA cloning (see Methods). e, f Left panels: Fold change in expression of circCHEK2 produced by minigenes with or without A-to-G mutations at three editing sties, upon knockdown of PTBP1 c or TDP43 d. Right panels: qPCR analysis showing the knockdown efficiency of PTBP1 and TDP43. g Left panel: Fold change in expression of endogenous circCHEK2 with or without lentivirus-mediated overexpression of ADAR1 in EC109 cells, upon knockdown of PTBP1. Middle panel: qPCR analysis showing the knockdown efficiency of PTBP1 in the indicated cells. Right panel: qPCR analysis illustrating the efficiency of ADAR1 overexpression in the indicated cells. b, c, e, f, g Each dot represents the mean of technical triplicates. Data are presented as mean ± S.D. of 3 biological replicates (paired, two-tailed Student’s t-test. n.s., not significant; *P < 0.05; **P < 0.01). h Number of editing-dependent ARcircs of which flanking introns have editing-mediated changes in the binding sites of each RBP. Black dots indicate RBPs included in this analysis and the number in the bracket denotes the number of circRNAs with altered binding motifs of the corresponding RBP due to editing in flanking introns. Those which have been previously reported to regulate circRNA biogenesis are highlighted in blue. PTBP1 is highlighted in red. Exact P values and source data are provided in Source Data file.
