Fig. 4: Analysis of SmD2-mediated alternative splicing in DNA repair genes.

a Differences in rMATS inclusion values in inducible shSNRPD2 HCCLM3 cells (n = 3/group biologically independent samples) after doxycycline (DOX) treatment are presented as median values, where the center line in each box represents the median, the edges of the boxes represent the first (25th percentile) and third (75th percentile) quartiles, and the whiskers extend to the minimum and maximum values. A5SS, alternative 5’ splicing site; A3SS, alternative 3’ splicing site; RI, retained introns; SE, skipped exons; MXE, mutually exclusive exons.” b A half-volcano plot showing SE events in DNA repair genes is highlighted, where an inclusion value difference < −0.01 and –log10(P-value) = 2 were used as thresholds, and events with y > 16 are shown as y = 16. c, Visualizations of RNA-seq profiles (sashimi plots) of DNA repair genes. d RT-PCR validation of alternative splicing events in BRCA1, FANCA, FANCD2, FANCG, and FANCI genes, with GADPH as a loading control. Numbers in white boxs indicate exon skipping. The experiment was repeated three times with similar results. e Immunoblot analysis of SmD2 depletion HCCLM3 cells transduced with shUPF1. The experiment was repeated three times with similar results. Half-life of BRCA1 transcripts with exon 9 skipping (f), FANCA transcripts with exon 37 skipping (g), and FANCD2 transcripts with exon 30 skipping (h) were measured by qPCR (n = 4 biologically independent samples). i qPCR examining the changes of spliceosomal snRNAs in HCCLM3 cells after DOX-induced SmD2 knockdown (n = 4 biologically independent samples). j U1 snRNA binding energy for 5’ splicing sites (5’ss) in skipped exons (n = 9580) influenced by SNRPD2 knockdown compared to constitutive exons (n = 200,234). The central line in each box indicates the median of the dataset, with the box edges representing the 25th and 75th percentiles. The whiskers span from the minimum to the maximum data points, without any points classified as outliers. k Inducible shSNRPD2 HCCLM3 cells were transfected with wild-type (WT) miniBRCA1 or with minigenes harboring mutations in the 5′ss, which were analyzed with RT–PCR assays to quantify the percent exon inclusion. Mutations that strengthen the 5′ss reduce the effects of SNRPD2 knockdown on miniBRCA1 SE. (ψ) Pseudouridine. The experiment was repeated three times with similar results. Mean values ± SD. Two-sided t-tests, **P = 0.002288, and ***P < 0.001. Source data are provided as a Source Data file.