Gain-of-function cardiomyopathic mutations in RBM20 rewire splicing regulation and re-distribute ribonucleoprotein granules within processing bodies

Abstract

Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic iPSCs with DCM-associated missense mutations in RBM20 as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulate contractile dysfunction of RBM20-associated DCM and reveal greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP reveals a gain-of-function preference of mutant RBM20 for 3' UTR sequences that are shared with amyotrophic lateral sclerosis (ALS) and processing-body associated RNA binding proteins (FUS, DDX6). Deep RNA sequencing reveals that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO. Super-resolution microscopy verifies that mutant RBM20 maintains very limited nuclear localization potential; rather, the mutant protein associates with cytoplasmic processing bodies (DDX6) under basal conditions, and with stress granules (G3BP1) following acute stress. Taken together, our results highlight a pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways.

Fig. 1. Generation and functional characterization of mutant iPSC-CMs.

a Targeted genomic region of RBM20. The location of R636 and the 8 nucleotides deleted in the 8-bp Del HMZ line are highlighted. b Genomic sequences of the generated RBM20 HTZ and HMZ iPSC lines. c Genomic sequence of the generated RBM20 8-bp Del HMZ line (KO). d iPSC-CM electrophysiological parameters from multi-electrode array (MEA) recordings. Color-coded paired replicates represent biologically independent experiments run in parallel. N = 3 biologically independent experiments. Data represented as mean values ± SEM. e Representative 3D-engineered heart tissues (3D-EHT) from WT and RBM20 mutant cell lines. Phase-contrast microscopy, scale bar: 1 mm. The location of flexible and glass-stiffened silicone posts is indicated. f Contractile metrics of 3D-EHTs. WT: n = 4, HTZ: n = 6, HMZ: n = 13, KO: n = 10. Data represented as mean values ± SEM. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test.

Fig. 2. Intracellular RBM20 localization in RBM20 Mutant iPSC-CMs.

a Immunofluorescence for RBM20 in WT and RBM20 mutant iPSC-CMs. Spinning disk confocal, scale bar: 5 μm. Representative micrographs from n > 3 experiments. b Structured illumination microscopy of DNA (magenta) and RBM20 (green). Left WT image represents X–Y maximum intensity projection, right images represent X–Z 3D projections. Yellow arrowheads indicate perinuclear localization of RBM20 in mutant cells. Yellow stars indicate the nuclear localization of RBM20. Scale bar: 5 μm. Representative micrographs from n > 3 experiments. c 3D localization analysis of RBM20 from 3D-SIM images. Magenta bars (left bar above genotype) indicate percent nuclear localization, green bars (right bar above genotype) indicate percent cytoplasmic localization. Data represents per-cell averages from N = 2 independent experiments with n = 4 cells per genotype. Data represented as mean values ± SEM.

Fig. 3. RBM20 mutant protein preferentially binds to the 3′ UTR of novel transcripts.

a Illustration of the eCLIP strategy for wild-type and R636S HMZ iPSC-CMs. b Frequency of reproducible eCLIP peaks for WT and R636S HMZ iPSC-CMs within coding sequence (cds) exons, introns and UTR regions. c HOMER de novo motif enrichment logos and hypergeometric (default) enrichment p-values for the top-ranked RNA-binding protein recognition elements defined from the CisBP-RNA database. RBM20-associated motifs are highlighted in red and RNA-stabilization-associated factors in blue. d Frequency bar chart of reproducible peaks in R636S HMZ eCLIP and the most similar eCLIP profiles from ENCODE (K562 or HepG2 cells), based on correlation to their frequency profiles. e Overlap of R636S peaks in reproducible ENCODE eCLIP peaks, ranked by their percentage overlap. One previously described ALS eCLIP profile for the FUS-P525L mutation is included and highlighted in red, representing peaks only found in the FUS mutant compared to controls. Blue text indicates RBPs with statistically enriched RBM20 motifs based on HOMER (Supplementary Fig. 4b). f Gene-set enrichment with GO-Elite (Fisher Exact test p < 0.05, raw) of genes associated with FUS-P525L and R636S overlapping peaks, for disease-associated gene-sets (red, DisGeNET), aggregate pathways (green, ToppFun), and Gene Ontology (black). g Overlap of genes up- or down-regulated by RNA-Seq in R636S iPSC-CMs (HTZ+HMZ) versus RBM20 deletion and R636S eCLIP peaks (eBayes two-sided t-test p < 0.05, FDR corrected). h Heatmap of all statistically ranked and organized (MarkerFinder algorithm) RBM20 R636S or deletion genes in iPSC-CMs by RNA-Seq. Statistically enriched gene-sets (GeneOntology + PathwayCommons) are indicated in blue with their associated Fisher’s Exact test p-value (unadjusted) and R636S eCLIP peaks indicated by a green dash (selected genes shown). Source data are provided as a Source Data file.

Fig. 4. 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 δPSI > 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, δPSI > 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.

Fig. 5. 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. 6. 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.