Fig. 1. Splice-regulatory activity of RBM20 variants is proportional to their nuclear localization.

a Immunofluorescence (IF) staining for RBM20 and alpha-actinin in iPSC-CMs, n = 3. b DAPI:RBM20 colocalization based on data from (a). Each dot represents a Pearson coefficient for at least five cells, n = 3. c ICS-based analysis of DRAQ5:RBM20 correlation in iPSC-CMs, n = 3. d qPCR analysis of TTN exon 242 splicing-out in iPSC-CMs normalized to GAPDH expression displayed as fold change versus the first replicate of RBM20-WT (means with standard errors, n = 3). e Left ventricular ejection fraction (LVEF) for patients with P633L (n = 4) or with other pathogenic or likely pathogenic (P/LP) mutations in the RSRSP stretch (n = 15) at diagnosis (norm for healthy individuals: 53 to 73%, green shading). Mean LVEF for P633L vs. P/LP variants: 52 ± 4.9% vs 43.1 ± 13.9%, P = 0.059 (two-sided T-test). f Initial LVEF as a function of the age at presentation (norm for healthy individuals: 53 to 73%, green shading, R2 = 0.001, P = 0.89, linear regression). g Internal Left Ventricular Diastolic Dimension (LVIDd) corrected for body size (norm for healthy individuals: 2.4–3.2 cm/m², green shading). Mean LVIDd/BSA for P633L (n = 3) vs. P/LP variants (n = 9): 3.2 ± 0.74% vs 2.8 ± 0.26%, P = 0.28 (two-sided T-test). h ICS-sorting strategy for RBM20-P633L, n = 3. i Numbers of differentially expressed genes (|log2FC| > 1 and P < 0.1, Wald test with Benjamini–Hochberg correction, DeSeq2⁵⁸) in pairwise comparisons to RBM20-WT, n = 3. j PSI values for alternative splicing events in the core RBM20 targets different between WT and R634Q (|delta PSI| > 0.1, P value < 0.05, two-sided T-test). k qPCR analysis of TTN exon 242 splicing-out in sorted iPSC-CMs, same representation as (d), two biological replicates, with two technical replicates each. l Enriched RBM20 interactors (FDR < 0.05 (Limma), and |log2FC| > 0.5 vs. no-bait control) in HeLa, n = 3. m Pathway enrichment analysis for common interactors between RBM20-WT, -P633L, and -R634Q in the nuclear fraction, Metascape⁵⁹. Boxplots (b, e, g) display quartiles Q1, Q2 (center), and Q3, with whiskers extending to the furthest data points within 1.5 times interquartile range (IQR). Statistical significance for (b, d, k) was calculated using one-way ANOVA with Tukey’s HSD post test (two-tailored): Ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Actual P values are shown in the source data file.