Fig 1: 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.
Fig 2: ADAR1-mediated editing of an ISS enhances SRSF7 binding for exon skipping.a Sequence chromatograms illustrate the editing level of the indicated sites (1–4) at intron 8 of CCDC15 pre-mRNA in HEK293T cells that were transfected with empty vector control (EV), ADAR1 (0.25, 1.0, or 2.0 µg), or ADAR2 (2.0 µg) expression construct. Black arrowhead indicates editing position. Red arrows show the location of primers used for PCR amplification. b Upper panel: schematic diagram of wild-type (WT) CCDC15 exon 8–9–10 minigene. The positions where an A-to-G mutation was introduced are highlighted in red (sites 1, 2, and 4) and purple (site 3). The 13-bp region deleted in the Del minigene is shaded in orange. Lower panel: RT-PCR analysis of exon 9 inclusion of exogenous CCDC15 transcripts in HEK293T cells that were transfected with the indicated WT or mutant minigenes (n = 3 or 2 biological replicates for each). 1 + 2 denotes both sites 1 and 2 were mutated from A to G. c In silico prediction of SRSF7 binding sites on the edited CCDC15 pre-mRNA by Human Splicing Finder (orange line) and RBPmap (blue line). The edited nucleotide at site 2 is highlighted in red. d RT-PCR analysis of exon 9 inclusion of exogenous CCDC15 transcripts in HEK293T cells that were co-transfected with WT or site 2-mutated (Mut 2) minigene together with EV or SRSF7 expression construct (n = 3 biological replicates for each). e WB analysis of RNA pulldown products (eluate) shows binding of SRSF7 and hnRNPK protein to the WT or Mut 2 RNA probes. Sequence of the Mut 2 probe is shown in c and the WT probe is the same except site 2 that remains as an unedited adenosine. FT, flow-through. b, d 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). Source data are provided as a Source Data file.
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