Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3
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PROVIDER: PRJNA911636 | ENA |
REPOSITORIES: ENA
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Project description:Severe forms of dilated cardiomyopathy (DCM) are associated with point mutations in the alternative splicing regulator RBM20 that are frequently located in the arginine/serine-rich domain (RS-domain). Such mutations can cause defective splicing and cytoplasmic mislocalization, which leads to the formation of detrimental cytoplasmic granules. Successful development of personalized therapies requires identifying the direct mechanisms of pathogenic RBM20 variants. Here, we decipher the molecular mechanism of RBM20 mislocalization and its specific role in DCM pathogenesis. We demonstrate that mislocalized RBM20 variants retain their splice regulatory activity, which reveals that aberrant cellular localization drives the pathological phenotype. A genome-wide CRISPR knock-out screen combined with image-enabled cell sorting identified Transportin-3 (TNPO3) as the main nuclear importer of RBM20. We show that the direct RBM20-TNPO3 interaction involves the RS-domain, disrupted by pathogenic variants. Re-localization of pathogenic RBM20 variants to the nucleus restores alternative splicing and dissolves cytoplasmic granules in cell culture and animal models. These findings provide proof-of-principle for developing therapeutic strategies to restore RBM20’s nuclear localization in RBM20-DCM patients.
2023-06-19 | PXD038790 | Pride
Project description:First, we compared gene expression and alternative splicing changes between differentially-localized P633L-RBM20, WT-RBM20, and R634Q-RBM20 in iPSC-CMs. Using ICS, we sorted iPSC-CMs with differentially-localized RBM20 based on correlation with nuclear staining. This was followed by RNA-sequencing of the sorted populations. Second, we analysed gene expression and splicing changes in iPSC-CMs overexpressing of WT-, R634Q-, or NLS-tagged R634Q-RBM20 in splice deficient cells carrying the homozygous frameshift mutation (S635FS). Third, we performed RNA sequencing of our HeLa Tet:Cas9 eGFP-RBM20-WT and -R634Q to compare their gene expression to iPSC-CMs. Lastly, we performed two genome-wide CRISPR ICS screens in HeLa cells stably expressing eGFP-RBM20-WT and -R634Q, and TetO-Cas9, to identify genes essential for RBM20 nuclear import.
2023-06-19 | GSE220833 | GEO
Project description:Mutations in RNA binding motif protein 20 (RBM20) are a common cause of dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine rich (RS-rich) domain, resulting in mis-localization of RBM20 to ribonucleoprotein granules within the cytoplasm, abnormal splicing of cardiac genes, and cardiomyocyte dysfunction. We used adenine base editing (ABE) and prime editing to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell-derived cardiomyocytes. We also created humanized Rbm20R636Q mutant mice, which succumbed to severe cardiac dysfunction, heart failure and premature death. Systemic delivery of ABE components by adeno-associated virus in these mice restored cardiac function and extended life span. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.
2022-08-11 | GSE210783 | GEO
Project description:Mutations in RNA binding motif protein 20 (RBM20) are a common cause of dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine rich (RS-rich) domain, resulting in mis-localization of RBM20 to ribonucleoprotein granules within the cytoplasm, abnormal splicing of cardiac genes, and cardiomyocyte dysfunction. We used adenine base editing (ABE) and prime editing to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell-derived cardiomyocytes. We also created humanized Rbm20R636Q mutant mice, which succumbed to severe cardiac dysfunction, heart failure and premature death. Systemic delivery of ABE components by adeno-associated virus in these mice restored cardiac function and extended life span. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.
2022-08-14 | GSE211005 | GEO
Project description:Human patients carrying genetic mutations in RBM20 develop a clinically aggressive dilated cardiomyopathy (DCM) characterized by early onset heart failure, high mortality, and sudden death, which is recapitulated in animal models. RBM20 has two primary domains, an RNA recognition motif (RRM) that binds RNA and an arginine/serine (RS)-rich domain that mediates spliceosome assembly and nuclear localization. Reported data showed that mutations in the RS domain lead to severe DCM. Loss of the RRM domain in RBM20 has been shown to disrupt the splicing of RBM20 target transcripts but does not lead to DCM. The objectives of the present study were to determine the functional role of the RS domain in DCM and examine the mechanisms. Mice expressing RBM20 lacking the RS domain (Rbm20ΔRS) were generated using CRISPR/Cas9 technology. Male and female Rbm20ΔRS mice developed a DCM-like phenotype characterized by ventricular dilation and impaired systolic function, that is more severe in females. Splicing of RBM20 target genes, including Ttn, was disrupted in both Rbm20ΔRS and Rbm20ΔRRM mice. However, RBM20 was mis-localized to the sarcoplasm only in the hearts of Rbm20ΔRS mice, indicating that mis-localization of RBM20 rather than disrupted splicing is key in DCM pathogenesis.
2023-01-02 | GSE212799 | GEO
Project description:Precise genomic editing of a pathogenic RBM20 mutation rescues dilated cardiomyopathy
| PRJNA867480 | ENA
Project description:Precise genomic editing of a pathogenic RBM20 mutation rescues dilated cardiomyopathy
| PRJNA868596 | ENA
Project description:Background: RBM20 gene is one of the genetic predispositions for dilated cardiomyopathy (DCM). Variants in RS domain has been reported in many DCM patients, while the pathogenicity of variants within the RNA-recognition motif remains unknown. Methods and results: We detected two human patients of I536T-RBM20 variant without DCM phenotype in sudden death cohorts. We performed splicing reporter assay and generated I538T knock-in (KI) mouse model (Rbm20I538T) in order to reveal the significance of this variant. The reporter assay revealed that human I536T variant affected TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice presented a different splicing pattern in Ttn, Ldb3, Camk2d and Ryr2. The expression of Casq1, Mybpc2 and Myot were upregulated in Rbm20I538T mice. Whereas, Rbm20I538T mice did show neither DCM nor cardiac dysfunction by histopathological examination and ultrasound echocardiography. Conclusion: I536T-RBM20 (I538T-Rbm20) variant does not cause DCM phenotype, but changes the gene splicing and expression effect. The splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d and Ryr2 may be contributory to sudden arrhythmogenic death.
2022-10-05 | GSE201018 | GEO
Project description:Deletion of the RS domain in RBM20 leads to dilated cardiomyopathy
| PRJNA877291 | ENA