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. 2022 Mar 21;13:1508. doi: 10.1038/s41467-022-29138-2

Fig. 3. ADAR1 binds and edits circCHEK2 RCM to promote circCHEK2 biogenesis.

Fig. 3

a Genome browser tracks of CHEK2 loci reveal ADAR1 binding peaks (top) from ADAR1-fRIPseq data and predicted RCM pairs within circCHEK2 flanking introns (bottom). Black arrow: the circular junction site of circCHEK2 in a 5’-3’ direction. Reads mapped to exonic or intronic regions (GENCODE annotation) are colored in red or blue, respectively. Potential match pairs are indicated in different colors and the pair with the highest BLAST score is defined as “Identified RCM”. b RIP-qPCR analysis of the association of ADAR1 protein to the circCHEK2 RCM region in EC109 cells transfected with FLAG empty vector (FLAG EV) or FLAG-ADAR1 (FLAG AR1). WB and qPCR analyses of FLAG-RIP immunoprecipitates are shown in the left and right panels, respectively. c Secondary structure formed by RCMs of circCHEK2, as predicted by RNAfold (left). Location of a reported ADAR1 binding motif is indicated by blue line. Blue arrows indicate 3 editing sites identified within RCMs of circCHEK2. Base-pair probabilities are shown by a color spectrum (middle). Pie charts illustrating the editing frequency (indicated by red slice) of each editing site in the indicated samples (right). Editing frequency of each editing site was measured using TA cloning (see Methods). d Schematic diagram illustrating the structure of circCHEK2 minigene. A 20-bp sequence of exon 3 was scrambled to distinguish minigene-produced transcripts from endogenous transcripts. e Fold change in expression of minigene-produced circCHEK2 and linear CHEK2, upon overexpression of WT or DeAD ADAR1, compared to EV control. f Fold change in expression between circCHEK2 and linear CHEK2 derived from the WT or mutated minigenes carrying A-to-G mutation(s) at editing sites. Edt1, A-to-G mutation at site #1; edt12, A-to-G mutations at sites #1 and #2, and so forth. b, e, f Data are presented as the mean ± S.D. of 3 biological replicates. Each dot represents the mean value of technical triplicates from an independent experiment. Data is presented as mean ± S.D. of 3 biological replicates. Statistical significance is determined by paired, two-tailed Student’s t-test (*, P < 0.05; n.s., not significant). Exact P values and source data are provided in Source Data file.