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

Abstract

RNA binding motif protein 20 (RBM20) is a key regulator of alternative splicing in the heart, and its mutation leads to malignant dilated cardiomyopathy (DCM). To understand the mechanism of RBM20-associated DCM, we engineered isogenic human induced pluripotent stem cells (iPSCs) with heterozygous or homozygous DCM-associated missense mutations in RBM20 (R636S) as well as RBM20 knockout (KO) iPSCs. iPSC-derived engineered heart tissues made from these cell lines recapitulated contractile dysfunction of RBM20-associated DCM and revealed greater dysfunction with missense mutations than KO. Analysis of RBM20 RNA binding by eCLIP revealed 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 revealed that the RBM20 R636S mutant has unique gene, splicing, polyadenylation and circular RNA defects that differ from RBM20 KO, impacting distinct cardiac signaling pathways. Splicing defects specific to KO or R636S mutations were supported by data from R636S gene-edited pig hearts and eCLIP. Super-resolution microscopy verified that mutant RBM20 maintains 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 novel pathogenic mechanism in cardiac disease through splicing-dependent and -independent pathways that are likely to mediate differential contractile phenotypes and stress-associated heart pathology.

Precise genomic editing of pathogenic mutations in RBM20 rescues dilated cardiomyopathy

“…Previous papers showed that aberrant RBM20 R636S granules colocalized with stress granules, marked by G3BP stress granule assembly factor 1 (G3BP1) in iPSC-CMs ( 13 , 15 ). Similarly, RBM20 R634Q granules also colocalized with stress granules in the cytoplasm after acute stress evoked by exposure to sodium arsenite (fig.…”

“…Therapeutic approaches using BE and PE have been applied for Duchenne muscular dystrophy, progeria, inheritable liver, and eye disorders in vivo (28,(31)(32)(33)(34)(35)(36)(37). The pathophysiology of RBM20 mutations is caused by a mixture of a loss of function and a pathogenic gain of function (11,13,15). Therefore, RBM20 mutations are potentially well suited for correction by precise genomic editing.…”

Transcription Factor GATA4 Regulates Cell Type–Specific Splicing Through Direct Interaction With RNA in Human Induced Pluripotent Stem Cell–Derived Cardiac Progenitors

“…Only a fraction of the GATA4-bound RNA were alternatively spliced, which may suggest GATA4 function in regulating other RNA-processing events, such as RNA polyadenylation or stability, similar to other RNA-binding proteins that regulate splicing including RBM20 (Figure 4A). 58 GATA4 was also bound to the gene body by chromatin immunoprecipitation sequencing at 28 of these 89 sites ( P <0.001), suggesting the possibility of cotranscriptional splicing regulation at some of these loci. The set of RNAs bound by GATA4 was enriched for genes involved in muscle stretch and calcium ion transport.…”

RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function

“…18 The cardiomyocyte-enriched RBM20 (RNA-binding motif 20) protein acts predominantly as a splicing repressor to exclude specific exons of several genes from mature mRNAs, including TTN . 19–22 Missense mutations in RBM20 cause familial DCM, features of which are recapitulated in genetically engineered pigs 22 and hiPSC-CMs (human induced pluripotent stem cell-derived cardiomyocytes). 19 Of note, in addition to defects in pre-mRNA processing, RBM20 mutations also induce the accumulation of sarcoplasmic ribonucleoprotein granules, which are likely to contribute to disease pathogenesis in a pleiotropic fashion.…”

“…19 Of note, in addition to defects in pre-mRNA processing, RBM20 mutations also induce the accumulation of sarcoplasmic ribonucleoprotein granules, which are likely to contribute to disease pathogenesis in a pleiotropic fashion. 19,22…”