Fig 1: Involvement of GA-rich motif(s) in ADARs-mediated splicing regulation.a, b The top four motifs enriched in ADARs-regulated cassette exons a and non-ADARs-regulated cassette exons b as provided by MEME. Height of letter depicts occurrence of nucleotides at specific position. E-value represents statistical significance of the motif. Target represents the number of cassette exons containing a particular motif over total input number. c RNA secondary structure predictions for pre-mRNA sequences of representative ADARs-regulated cassette exons by RNAfold. Base-pair probabilities are shown by a color spectrum. Red line indicates the GA-rich motif. Asterisk denotes editing site. d Sequence chromatograms illustrate editing site adjacent to GA-rich motif of AKAP11 and KLRG1 upon ADAR2 overexpression in HEK293T cells. GA-rich motif is shaded in orange. Black arrowhead indicates editing position.
Fig 2: Schematic mechanistic diagram of ADARs-regulated alternative splicing.Conversion of adenosine to inosine by ADAR1 at a GA-rich sequence proximal to cassette exon of a target transcript recruits SR proteins or other splicing factors to repress exon inclusion. Independent of their editing functions, ADAR1 and ADAR2 proteins can bind to inter-introns of a target transcript, thereby looping out the exon and blocking the access of the spliceosome to the splice site. Alternatively, ADAR2 blocks the access of U2AF65 to the 3′ splice site by binding to the dsRNA formed between the GA-rich sequence and Py-tract.
Fig 3: Preferential repressive and stimulative roles of DHX9 dependent on ADAR specificity. (A) Editing frequency of sites determined using Targeted-seq. Upper panel: Bar charts showing effects of ADARs OE on the indicated editing sites in EC109 cells. Changes in editing level are calculated by subtracting VAF of ADAR1 or ADAR2 OE with VAF of empty vector control (VAFADAR-VAFcontrol); Lower panel: Bar charts showing effects of DHX9 KD on the indicated editing sites in EC109 cells. Changes in editing frequency are calculated by using VAFshDHX9-VAFshScr. Data represented as mean ± s.e.m. Experiment was performed with two biological replicates. VAF, variant allele frequency. (B) Representative Sanger sequencing chromatograms validate ADAR substrate specificity of two representative ADAR1- or ADAR2-specific sites. Percentage represents the editing frequency calculated by taking the peak area of ‘G’ peak over sum of ‘A’ and ‘G’ peaks.
Fig 4: Domains involved in ADARs/DHX9 interaction. (A) Domain mapping of ADAR1 supporting ADAR1–DHX9 interaction. Lysates harvested from EC109 cells co-transfected with DHX9-Flag and ADAR1-V5 wild-type or truncation mutants were used for IP with anti-V5 antibodies. (B) Mapping of the ADAR2 domain required for ADAR2–DHX9 interaction. Lysates harvested from EC109 cells co-transfected with DHX9-Flag and ADAR2-V5 wild-type or truncation mutants were used for IP with anti-V5 antibodies. (C) Mapping of the DHX9 domain required for ADARs–DHX9 interaction. Lysates harvested from EC109 cells co-transfected with ADAR1-Flag or ADAR2-Flag and DHX9-V5 wild-type or truncation mutants were used for IP with anti-V5 antibodies. (D) Schematic diagrams of full-length ADAR1, ADAR2 and DHX9, with their truncation mutants. The extent of interaction is indicated as positive (+) and negative (−). dsRBD, double-stranded RNA binding domain; MTAD, minimal transactivation domain; HelC, helicase domain. (E) Co-IP of DHX9-V5 with wild-type or dsRNA-binding deficient (EAA) ADAR mutants. For each sample, 5% of the total cell lysates used in the IP reaction was loaded as an input control (Figure 2A–C and E).
Fig 5: ADARs repress exon inclusion editing dependently and independently.a RT-PCR analysis of CCDC15-ex9 inclusion in HEK293T (left) and EC109 (right) cells that were transfected with the indicated wild-type ADAR1 (AR1) or ADAR2 (AR2) and different mutant (EAA and DeAD) forms of expression constructs (n = 4 biological replicates for each). EV, empty vector. b RT-PCR analysis of CCDC15-ex9 inclusion in HEK293T that were transfected with the indicated amount of EV, ADAR1, or ADAR2 construct (n = 3 biological replicates for each). Statistical significance of dose treatment is determined by linear regression. c RT-PCR analysis of RELL2-ex3 inclusion in the same samples as described in a (n = 3 or 4 biological replicates for each). a, c 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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