Fig. 5: ESRP1 is the key regulator of splicing diversity in shaping TPIT heterogeneity.

a Bubble heatmap showing the top 10 dysregulated RBPs in the TPIT lineage. b UMAP plots (left) showing cells from TPIT lineage. The colors represent different subtypes. The scatter plot (right) shows the expression of ESRP1 between the silent and functional ACTH clusters. c Heatmap displaying the number of detected ESRP1 motifs in each TPIT-lineage-specific splicing event validated by single-cell RNA-seq analysis. The ESRP1 motif (upper) is collected from the database (RBPmap, http://rbpmap.technion.ac.il/). The top enriched motif calculated from the differentially spliced events is displayed in the lower panel. d Venn diagram showing the overlap between events differentially spliced after ESRP1 knockdown and overexpression (RNA-seq data) in primary cells from PitNET patients. The P-values are calculated by the one-sided hypergeometric test. e–g Scatter plots showing the expression of ESRP1 (y-axis) versus the PSI of ARFGAP2 (x-axis) (e), ENAH (x-axis) (f), and ARFGAP1 (x-axis) (g) in 58 patients of TPIT lineage. The colors represent different subtypes. The P-values are calculated by a two-sided Spearman’s correlation analysis. h qRT-PCR showing the relative mRNA expression of ESRP1 normalized to GAPDH in patients from the functional (n = 5) and silent ACTH subtypes (n = 6). The P-values are calculated by a two-sided unpaired Student’s t-test. Data are presented as mean values ± SD. i RT-PCR validation of the effect of ESRP1 knockdown on the TPIT lineage-specific splicing events in primary TPIT patient samples. Changes in splicing events under the condition of two ESRP1 siRNA treatments are measured by RT-PCR using gene-specific primers (sequences listed in Supplementary Data S14). Data are presented as the mean ± SD of three independent replicates. The P-values are calculated by a two-sided unpaired Student’s t-test. Source data are provided as a Source Data file.