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Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs.

ABSTRACT: Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

SUBMITTER: Schneider JW

PROVIDER: S-EPMC9270981 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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.

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Project description:Background Variants in the cardiomyocyte-specific RNA splicing factor RBM20 have been linked to familial cardiomyopathy, but the causative genetic architecture and clinical consequences of this disease are incompletely defined. Methods and Results To define the genetic architecture of RBM20 cardiomyopathy, we first established a database of RBM20 variants associated with cardiomyopathy and compared these to variants observed in the general population with respect to their location in the RBM20 coding transcript. We identified 2 regions significantly enriched for cardiomyopathy-associated variants in exons 9 and 11. We then assembled a registry of 74 patients with RBM20 variants from 8 institutions across the world (44 index cases and 30 from cascade testing). This RBM20 patient registry revealed highly prevalent family history of sudden cardiac death (51%) and cardiomyopathy (72%) among index cases and a high prevalence of composite arrhythmias (including atrial fibrillation, nonsustained ventricular tachycardia, implantable cardiac defibrillator discharge, and sudden cardiac arrest, 43%). Patients harboring variants in cardiomyopathy-enriched regions identified by our variant database analysis were enriched for these findings. Further, these characteristics were more prevalent in the RBM20 registry than in large cohorts of patients with dilated cardiomyopathy and TTNtv cardiomyopathy and not significantly different from a cohort of patients with LMNA-associated cardiomyopathy. Conclusions Our data establish RBM20 cardiomyopathy as a highly penetrant and arrhythmogenic cardiomyopathy. These findings underline the importance of arrhythmia surveillance and family screening in this disease and represent the first step in defining the genetic architecture of RBM20 disease causality on a population level.

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Project description:Transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN) genome editing technology enables site directed engineering of the genome. Here we demonstrate for the first time that both TALEN and ZFN injected directly into pig zygotes can produce live genome edited pigs. Monoallelic as well as heterozygous and homozygous biallelic events were identified, significantly broadening the use of genome editor technology in livestock by enabling gene knockout in zygotes from any chosen mating.

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