Fig 2: DAP3 functions as a potent editing repressor in ESCC cells.(A) WB analysis of the indicated proteins in stable DAP3-knockdown EC109 and KYSE180 cells. β-Actin (Actin) was used as a loading control. (B) Distribution of DAP3-affected editing sites over annotated genomic regions in EC109 and KYSE180 cells. (C) Venn diagram showing genes containing DAP3-affected sites in EC109 and KYSE180 cells. (D) Transcriptome-wide RNA editing analysis uncovers DAP3-knockdown–mediated changes in A-to-I editome of EC109 and KYSE180 cells. Changes in editing level are calculated by subtracting VAF (variant allele frequency) of shDAP3 with VAF of shscr (VAFshDAP3 − VAFshscr). Data are shown as the average editing change (mean; deep blue) induced by DAP3 sh1 and sh2 with the SE (SEM; light blue). (E) Sanger sequencing chromatograms illustrate editing of randomly selected sites (top). Quantification of editing frequency of each site is shown in the bottom. Data are presented as mean ± SEM. of technical triplicates. (F) Quantification of editing frequency of DAP3-affected sites in WT and DAP3-KO EC109 cells. Data are presented as mean ± SEM. of three individual clones generated by the CRISPR-Cas9 method. (E and F) Percentage represents the editing frequency calculated by taking the peak area of “G” peak over the sum of “A” and “G” peaks. Arrow indicates position of editing. Statistical analysis is conducted using unpaired, two-tailed Student’s t test (*P < 0.05; **P < 0.01).

Fig 3: DAP3 increases WSB1 expression via suppressing AS-NMD of WSB1 to promote tumorigenesis.a Schematic diagram illustrating the inclusion of non-canonical exon E6a and E6b introduces a PTC to WSB1 mRNA transcript, which may result in NMD. b Semiquantitative RT-PCR analysis of the WSB1 splicing upon inhibition of NMD. Left panel: EC109 cells were treated with CHX or vehicle control (DMSO) for 6 h. Right panel: EC109 cells were transfected with shUPF1 or the scramble control (Scr) for 48 h. SRSF1 or ZDHHC16 serves as a positive or negative control, respectively, to ensure successful inhibition of NMD. ACTB was used as a housekeeping gene internal control. Data are represented as mean ± s.d. of n = 3 biologically independent samples. c qRT-PCR analysis of the change in expression of WSB1 canonical isoform (E6a/6b-skipped) upon DAP3-KD (upper panel) or KO (lower panel). ACTB was used as a housekeeping gene internal control. Data are represented as mean ± s.d. of technical triplicates. d–f Western blot analyses of DAP3, WSB1, ATM, and ACTB protein expression in d DAP3-KD EC109 cells, e DAP3-KO EC109 cells (“1” and “2” indicate two different WT or KO clones), and f EC109 cells with DAP3 overexpression. EV empty vector control. g Western blot analysis of DAP3 and WSB1 protein expression in DAP3-KO or WT EC109 cells that were overexpressed with the empty vector control or WSB1 construct. h, i Quantification of foci formation (h) or soft agar colony formation (i) induced by the indicated stable cells. Scale bar: 200μm. Data are represented as mean ± s.d. of n = 3 biologically independent wells. b, c, h, i Statistical significance is determined by unpaired, two-tailed Student’s t-test (*p < 0.05, **p < 0.01). j Xenograft tumors derived from the indicated stable cell lines at end point (n = 6 mice per group). k Growth curve of tumors derived from the indicated cells in mice, over a 17-day observation period. Data are presented as the mean ± s.e.m. Statistical significance is determined by unpaired, two-tailed Student’s t-test (*p < 0.05). Exact p-values and source data are provided in Source Data file.

Fig 4: DAP3 depletion leads to widespread splicing changes in cancer cells.a Pie charts showing the distribution of each type of significantly altered splicing events in DAP3-depleted EC109 and KYSE180 cells compared to the scramble controls detected by rMATS15. SE skipped exon, MXE mutually exclusive exons, A5SS alternative 5’ splice site, A3SS alternative 3’ splice site, IR intron retention. b Percentage of DAP3-modulated splicing events demonstrating increased or decreased PSI upon DAP3 depletion in EC109 and KYSE180 cells. c Venn diagram showing the numbers of genes which underwent DAP3-modulated splicing and genes containing DAP3 eCLIP peaks in EC109 cells. Bar chart showing the percentage of genes with or without DAP3-modulated splicing that are bound by DAP3 (two-sided Fisher’s Test). d The binned DAP3 eCLIP peak coverage across the splice junctions of five types of DAP3-modulated splicing events in EC109 cells. e Western blot analysis of DAP3 protein expression in the indicated DAP3 knockdown (KD, sh1, and sh2), knockout (KO) and their control [scramble (Scr) and wildtype (WT)] samples. f, g Semiquantitative RT-PCR analyses of five randomly selected DAP3-modulated splicing events. h IGV browser tracks of the DAP3 eCLIP peaks spanning exon 5–6 and intervening introns of WSB1 gene. Significant peaks are marked by blue and green bars. Schematic diagram illustrates the genomic fragments inserted into the wildtype (wt) and mutant WSB1 splicing minigenes. del mut1, lacking a 58 bp DAP3-binding sequence in exon E6a/b; del mut2, lacking a 9 bp DAP3-binding motif in exon E6a/b. i Semiquantitative RT-PCR analyses of splicing changes of exogenous WSB1 transcripts derived from the indicated minigenes in Scr control and DAP3-KD cells. j RNA pulldown assay detecting the binding of DAP3 to WSB1 exon E6b wt, del mut1, and del mut2 RNA probes. WB analysis of DAP3 proteins in RNA pulldown (eluate) products and flow-through (FT) fractions. f, g, i Data are represented as mean ± s.d. of n = 3 biologically independent samples. Statistical significance is determined by unpaired, two-tailed Student’s t-test (*p < 0.05, **p < 0.01). Exact p-values and source data are provided in Source Data file.

Fig 5: RNA-binding landscape of DAP3 by eCLIP-Seq.a The distribution of the DAP3 eCLIP peaks in the human genome from biological duplicates (DAP3-1 and DAP3-2). TSS transcription start site, TTS transcription termination site, UTR untranslated region. b The binned DAP3 eCLIP peak coverage across all expressed genes in EC109 cells. The 5’UTR, CDS, and 3’UTR of each gene are split into 13, 100, and 70 bins, respectively. CDS coding sequence. c Integrated genome viewer (IGV) browser tracks of the DAP3 eCLIP peaks spanning the genomic loci of NEAT1 and MALAT1, 5’ region of ARHGEF16, and 3’ region of TARDBP gene. Detailed information about all significantly enriched eCLIP peaks can be found in Supplementary data 1. d Top three most significantly enriched de novo sequence motifs in the DAP3-binding peaks using HOMER12. The percentage of peaks containing the discovered motifs and the p-values of the motifs calculated by binomial test against the random genomic background were shown. e REMSA analysis of the binding of DAP3 to two RNA probes that were generated from DAP3-binding sequences within SCYL1 and ATAD3A transcripts. A 100-fold molar excess of nonbiotinylated RNA probes was used as competitor. f Gene ontology analysis of DAP3-bound genes. The top 10 most significantly enriched biological processes are shown. The significance of enrichment for GO sets were evaluated by the WebGestalt39 portal default hypergeometric test. Source data are provided in Source Data file.