Fig 1: PUS7 regulates pseudouridine (Ψ) levels on nuclear RNA.A Differential expression of PUS7 in normal and tumor colon tissues from the TCGA GTEx dataset. p value was analyzed by two-tailed Student’s t test. B Cell proliferation in the colon cancer cell line HCT116 with or without PUS7 knockdown. Data are presented as mean ± SEM (n = 3, biological replicates), analyzed by two-tailed Student’s t test. C Cell proliferation in the colon cancer cell line DLD-1 with or without PUS7 knockdown. Data are presented as mean ± SEM (n = 3, biological replicates), analyzed by two-tailed Student’s t test. D Western blot analysis of PUS7 in cytosolic, nucleoplasmic, and chromatin fractions, with β-tubulin (cytoplasmic), SNRP70 (nucleoplasmic), and H3 (chromatin) as controls. Cell line used: HCT116. This experiment was repeated independently twice with similar results. E Change in Ψ levels on mRNA in HCT116 cells with or without PUS7 knockdown, quantified by Triple Quad LC-MS/MS. Data are presented as mean ± SEM (n = 3, biological replicates), analyzed by two-tailed Student’s t test. F Change in Ψ levels on nucleus RNA (<200 nt, ribosomal RNA-depleted) in HCT116 cells with or without PUS7 knockdown, quantified by Triple Quad LC-MS/MS. Data are presented as mean ± SEM (n = 3, biological replicates), analyzed by two-tailed Student’s t test. G Change in Ψ levels on nuclear RNA (>200 nt, ribosomal RNA-depleted) in HCT116 cells with or without PUS7 knockdown, quantified by Triple Quad LC-MS/MS. Data are presented as mean ± SEM (n = 3, biological replicates), analyzed by two-tailed Student’s t test. H Ψ detection workflow using bisulfite-induced deletion sequencing (BID-seq). I Distribution of pseudouridine sites on nuclear RNA in HCT116 cells. J Heatmap showing differentially regulated Ψ sites. K Heatmap of 8 differentially regulated Ψ sites containing the UGUAG motif, a potential PUS7 substrate motif. p value significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.