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.
Highlights
Mutations in the cardiac splicing factor RNA-binding motif protein 20 (RBM20) lead to malignant dilated cardiomyopathy (DCM)
The heterozygous mutation (R636S HTZ) was introduced into induced pluripotent stem cells (iPSCs) derived from a healthy male subject (WTC11) using transcription activator-like effector nucleases (TALENs) and a droplet digital PCR based strategy (Fig. 1a, b)[25,26]
We applied precision targeting of an RBM20 patient genetic mutation in human iPSC-CMs as a model system to evaluate the impact of specific patient alleles and RBM20 KO
Summary
Mutations in the cardiac splicing factor RBM20 lead to malignant dilated cardiomyopathy (DCM). In an impressive recent effort, pigs were engineered with either heterozygous (HTZ) or homozygous (HMZ) R636S mutations in RBM2024 This model led to three crucial observations: (1) RBM20 R636S HMZ mutation leads to highly penetrant neonatal lethality due to heart failure; (2) mutant RBM20 co-localizes with stress granules in the CM cytoplasm after metabolic stress induced by sodium arsenate; and (3) RBM20 undergoes an apparent liquid–liquid phase separation. These studies raised intriguing questions regarding the role of RBM20 in pathogenesis, physiology, normal cardiac development, and downstream RNA biology
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