Fig 1: Genome-wide ICS screen identifies TNPO3 as the main nuclear transporter of RBM20.a Schematic outline of the ICS screen. Six genome-wide libraries were applied to HeLa cells expressing eGFP-RBM20-WT and Tet::Cas9, with 100 cells per gRNA coverage. Cells were sorted based on the correlation between RBM20 and DRAQ5 into 7% higher and 7% lower fractions at final coverage of 500 cells per gRNA per sorted bin. Unsorted input samples were collected too. b Reads were combined in silico to one dataset, and hits were called using MAUDE69. Genes are ranked by their statistical significance. The horizontal dashed lines indicate an FDR of 1%. Positive/negative regulators with FDR < 1% are marked in red and blue, respectively. c Scatter plot of fold changes visualizing gRNA abundance changes in higher (x axis) and lower (y axis) sorted bins compared with the plasmid library. Red and blue dots indicate statistically significant positive and negative regulators, respectively (FDR < 5% according to MAUDE). Labeled are positive regulators selected for future analyses. d The impact of single knockouts of the selected hits (one gRNA per gene picked based on the strongest Z-score from the pooled screen) on RBM20 localization tested with ICS. The top row in the heatmap shows the log10(FDR) value for each candidate from the screen. The phenotype in the second row represents the standardized difference in RBM20 localization between the knockout (KO) and control cell populations (log2 of the ratio between cell fraction with Pearson coefficient (DRAQ5:RBM20) > 0.7 in the KO divided by cell fraction with Pearson coefficient (DRAQ5:RBM20) >0.7 in the control). e DAPI:RBM20 correlation quantified based on fluorescence microscopy analysis shown in Supplementary Fig. 5g, for the single KOs indicated. Each dot represents a Pearson correlation coefficient R for at least five cells, n = 3. Boxplots display quartiles Q1, Q2 (center), and Q3, with whiskers extending to the furthest data point within 1.5 times the IQR. Ns not significant, ***P < 0.001, one-way ANOVA with Tukey’s HSD post test (two-sided). Actual P values are shown in the source data file.
Fig 2: Model of mutant RBM20 differential splicing and P-body impacts in dilated cardiomyopathy.Proposed model for the impact of wild-type and mutant RBM20 on nuclear regulation of splicing, based on RNA-Seq and eCLIP data, as compared to the cytoplasmic role of mutant RBM20 on P-body formation and 3'UTR association with mRNAs implicated in granule formation.
Fig 3: Mislocalization of RS-domain RBM20 mutants is caused by loss of interaction with TNPO3.a ICS-measured DRAQ5:RBM20 correlation in WT or P633L iPSC-CMs transfected with control (Ctr) or TNPO3 siRNA. b DAPI:RBM20 correlation (based on Supplementary Fig. 7a). Each dot represents a Pearson coefficient for at least five cells, n = 3. c qPCR analysis of TTN exon 242 splicing-out in WT or P633L iPSC-CMs with Ctr or TNPO3 siRNA normalized to GAPDH (mean fold change versus the RBM20-WT with control siRNA, with standard errors, two biological replicates with two technical replicates each). d ICS-measured DRAQ5:RBM20 correlation for HeLa expressing eGFP-WT-, -P633L-, -R634Q-, or -RSS-RBM20, with Ctr or TNPO3 siRNA. e DAPI:RBM20 correlation (based on Supplementary Fig. 8a). Each dot represents a Pearson coefficient for at least five cells, n = 3. f Superimposed AlphaFold2 models of the RRM-RS domain of RBM20 (amino acid 511–673) as WT (gray), P633L (light blue), or R634Q (salmon). g Representative AlphaFold2 model of RBM20’s WT RRM-RS domain (blue, amino acid 511–673) in complex with TNPO3 (gray, full-length, amino acid 1-923). The PRSRSP stretch (amino acid 633–638) in RBM20 is highlighted as red spheres. h Predicted changes in binding affinity between TNPO3 and RBM20 upon RS-domain mutations. N = 20 AlphaFold models of the wild-type RBM20-TNPO3 complex were used to calculate the affinity change with a point mutation (see “Methods”). i Quantification of TNPO3 co-immunoprecipitating with RBM20 based on western blot analysis, representative image is displayed in (j) (n = 3, means with standard errors). j Western blot analysis of RBM20, TNPO3, and MOV10 in the cytoplasmic fraction of HeLa, and their co-immunoprecipitation with eGFP-RBM20 (Neg = negative no-bait control). k Quantification of TNPO3 peptides identified by mass spectrometry in the cytoplasmic fractions of co-immunoprecipitants with indicated cells, normalized to Neg, n = 3. Boxplots (b, e, h, k) display quartiles Q1, Q2 (center), and Q3, with whiskers extending to the furthest data point within 1.5 times the IQR. Ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t-test for (b), and one-way ANOVA with Tukey’s HSD post test for (c), (e), all two-tailored. Actual P values are shown in the source data file.
Fig 4: Intracellular localization of RBM20 mutant iPSC-CMs and association with P-bodies.a Localization of RBM20 (green) and DDX6 (processing bodies, magenta). High-magnification insets highlight co-localization. Spinning disk confocal, scale bar: 5 µm. Representative micrographs from n > 3 independent biological experiments. b DDX6/RBM20 co-localization is quantified via Pearson's coefficient of co-localization. Data from N = 3 biological independent experiments. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. Data represented as mean values ± SEM. c Structured Illumination microscopy of DDX6 (left), RBM20 (middle), and merged channels (DDX6: magenta, RBM20: green). White arrows indicate co-localization; yellow asterisk indicates RBM20 not associated with processing bodies. Scale bar: 0.5 µm. Representative micrographs from n = 2 independent biological experiments. d Assessment of stress granules via G3BP1 localization. iPSC-CMs were analyzed in basal conditions (e.g., normal media) and in response to stress (1 mM sodium Arsenate treatment for 1 h). Spinning disk confocal, scale bar: 5 µm. Representative micrographs from n > 3 independent biological experiments. e Structured illumination microscopy analysis of RBM20 (yellow) and G3BP1 stress granules (cyan) in sodium-arsenate-treated iPSC-CMs. Scale bar: 5 µm. Representative micrographs from n = 2 independent biological experiments. Source data are provided as a Source Data file.
Fig 5: RBM20 mutation and knockout impact distinct pathways at the level of alternative splicing.a Percentage of alternative splicing events considered differential (LIMMA t-test p < 0.1, FDR corrected and dPSI > 0.1) for each of the iPSC-CM genotypes versus WT, separated by the predicted event-type (e.g., cassette-exon, intron retention) (top). Below, the percentage of splicing events associated with either cassette-exon inclusion or exclusion (skipping) are shown for each RBM20 genotype vs. WT. b Gene-level visualization of exon-level relative expression levels (splicing-index) for two of example significant genes, TTN and CDC14B, predicted to be alternatively spliced in R636S HTZ and HMZ cells in a dosage-dependent manner. AltAnalyze exon identifiers are shown below. c Heatmap of the predominant alternative splicing patterns (MarkerFinder) for all reasonably detected splicing events (eBayes two-sided t-test p < 0.05, dPSI > 0.1). The description of each pattern is displayed to the left of the heatmap (Incl exon-inclusion, Excl exon-exclusion associated splicing events). Splicing events with intronic eCLIP peaks in the same gene or that are also observed for the same exon–exon junctions as neonatal R636S porcine model (orthologous genome coordinates) are denoted to the right of the plot with examples listed. Underlined splicing-event genes indicate prior evidence of RBM20-dependent alternative splicing from rat KO studies. Associated statistics for all displayed events are provided in Supplementary Data 14–20. d–i SashimiPlot genome visualization of RBM20-dependent splicing events observed in iPSC-CMs, associating with distinct patterns of regulation. Representative samples were selected. Specifically, R636S-allele dosage-dependent splicing (d), dosage-independent R636S splicing (e), R636S but not KO-dependent splicing (f), R636S-HTZ-specific events (g), R636S-HMZ-specific events, and RBM20-KO specific events (i) among a series of those visualized by SashimiPlot analysis (see Supplementary Fig. 5 and S6). Splice-junction read counts are denoted above each curved exon–exon junction line, along with the estimated percentage of exon-inclusion. j Gene-set enrichment analysis (Fisher’s Exact test p-value, unadjusted) of splicing-events segregated according to the MarkerFinder assigned patterns (panel c). Gene-sets correspond to either Mouse Phenotype Ontology or a collection of Pathway databases from ToppCell. *Verified splicing event patterns inferred from independently edited iPSC-CMs. °Verified splicing event from R636S HTZ edited pig hearts. • R636S eCLIP intron bound peak containing. Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-RBM20 antibody