Fig. 4: Inter-intronic dsRNA is essential for ADAR1/2 binding and splicing regulation.

a Schematic diagram illustrates serial deletions introduced into intron 9. Red asterisk represents editing site. b RT-PCR analysis of exon 9 inclusion of exogenous CCDC15 transcripts in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct (n = 3 or 2 biological replicates for each). Data are presented as the mean ± S.D. of percent spliced in (PSI) values from biological replicates. Each dot represents a biological replicate. Statistical significance is determined by paired t-test (*P < 0.05; **P < 0.01). c Sequence chromatograms illustrate the editing level of the indicated sites (1–4) in HEK293T cells that were co-transfected with the indicated minigene and overexpression construct. Black arrowhead indicates editing position. d In silico prediction of RNA secondary structure by RNAfold. Minimum free energy structures drawing encoding base-pair probabilities are shown. Base-pair probabilities are shown by a color spectrum. Arrow indicates the editing site. e REMSA analysis of binding of ADAR1 or ADAR2 protein to CCDC15 transcripts in vitro, using a ³²P-labeled RNA probe which simulates the dsRNA formed between introns 8 and 9 (CCDC15 In8-9 WT) together with the increasing amount of recombinant ADAR1/2 protein. f RIP-quantitative PCR (qPCR) analysis of the binding of ADAR1 or ADAR2 protein to exogenous CCDC15 transcripts (edited region in intron 8 and ECS in intron 9) in vivo (bottom panel). HEK293T cells were transfected with FLAG empty vector, FLAG-ADAR1, or FLAG-ADAR2, together with the wild-type CCDC15 minigene, followed by RIP assay at 48 h post transfection. WB analysis of FLAG-RIP immunoprecipitates is shown in the top panel. Input indicates 1% of the total cell lysate. Data is presented as mean ± S.D. of %input derived from qPCR technical triplicates from a representative experiment. Each dot represents a technical replicate. Statistical significance is determined by unpaired, two-tailed Student’s t-test (***P < 0.001). g REMSA analysis of the binding of SRSF7 to CCDC15 transcripts in the absence or presence of ADAR1 protein in vitro, using a ³²P-labeled CCDC15 In8-9 WT long dsRNA probe. Source data are provided as a Source Data file.