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.

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

“…[19][20][21][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. 19,22 Newborns with hypoplastic left heart syndrome (HLHS) were found to carry de novo mutations in the RBP gene RBFOX2 (RNA binding Fox-1 homolog 2).…”

“…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][20][21][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 Newborns with hypoplastic left heart syndrome (HLHS) were found to carry de novo mutations in the RBP gene RBFOX2 (RNA binding Fox-1 homolog 2). 23 Cardiomyocyte-specific ablation of RBFOX2 in the mouse embryo induced a structural heart defect with some features of HLHS.…”

“… 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 …”

The grand challenge of discovering new cardiovascular drugs

“…Likewise, a thorough molecular and mechanistic understanding of the pathogenesis of rare inherited dilated cardiomyopathies promises to reveal new therapeutic insights. For example, future therapies for heart failure due to RBM20 , LMNA , and BAG3 mutations may involve specific strategies that modulate RNA metabolism ( Guo et al, 2012 ; Green et al, 2016 ; Schneider et al, 2020 ; Fenix et al, 2021 ), mechanosensing ( Chai et al, 2021 ) and proteostasis ( Meister-Broekema et al, 2018 ; Ding et al, 2019 ; Martin et al, 2021 ), respectively.…”