Fig. 7: The Y65C mutation results in the proximal PAS-biased APA pattern.
a Venn diagram delineating the overlapping distribution of differential binding proteins across parallel IP-MS datasets. n = 3 mice in each group in each independent experiment. b Top 10 enriched biological processes ranked by P-value. Analysis of the overlapping proteins from (a) using a one-sided hypergeometric test with Benjamini–Hochberg FDR correction. Numbers and the size of circles represent counts. c Heatmap visualization reveals differential expression patterns of enhanced binding proteins from (a). IBAQ intensities from duplicate IP-MS experiments were quantile-normalized and averaged. d Scatter plot depicting the genes with APA changes (FDR < 0.05) in E12.5 Pqbp1Y65C/Y cortex. Blue: Transcripts with proximal PAS preference in Y65C. Red: Transcripts demonstrating distal PAS preference in Y65C. n = 3 mice in each group. e The genes with APA changes (FDR < 0.05) in E12.5 Pqbp1-cKO cortex. Blue: transcripts with proximal PAS preference in cKO compared to the control; Red: Transcripts demonstrating distal PAS preference in cKO. n = 3 mice in each group. f Venn diagram shows the overlapped targets between Pqbp1Y65C/Y and Pqbp1-cKO. g The genes overlaping with known ESC self-renewal regulators. h Integrative Genomics Viewer tracks show the representative gene reads of Marcksl1 from RNA-seq. i qRT-PCR measures the ratios of transcripts with the extended 3′ UTR of Marcksl1. The locations of primer sets used to amplify specific regions are marked (top). n = 3 mice in each group, p = 0.0329. j CoIP of PQBP1 and FIP1L1. The cortex of mice at E15.5 was immunoprecipitated with anti-PQBP1 antibodies. n = 3 biologically independent experiments, p = 0.0085. k, l Dual-luciferase reporter assays for Flag, Flag-PQBP1, and Flag-PQBP1Y65C groups. The ratios of R/F (Renilla luciferase 480 nm/firefly luciferase 560 nm) were normalized to the Flag group. CMV, CMV promoter; R luc, Renilla luciferase gene; IRES, internal ribosomal entry site; F luc, firefly luciferase gene. n = 5 biologically independent experiments, Flag vs Flag PQBP1: p < 0.0001; Flag vs Flag PQBP1Y65C: p = 0.0002. All quantification data are represented as mean ± SD. j Two-tailed unpaired Student’s t test. l Two-tailed one-way ANOVA with Tukey’s multiple comparisons test to adjust for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Source data are provided as a Source Data file.
