Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy
Yanghai Zhang, Zachery R. Gregorich, Yujuan Wang, Camila Urbano Braz, Jibin Zhang, Yang Liu, Peiheng Liu, Jiaxi Shen, Nanyumuzi Aori, Timothy A. Hacker, Henk Granzier, Wei Guo
Yanghai Zhang, Zachery R. Gregorich, Yujuan Wang, Camila Urbano Braz, Jibin Zhang, Yang Liu, Peiheng Liu, Jiaxi Shen, Nanyumuzi Aori, Timothy A. Hacker, Henk Granzier, Wei Guo
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Research Article Cardiology Cell biology

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy

Abstract

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain–mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

Authors

Yanghai Zhang, Zachery R. Gregorich, Yujuan Wang, Camila Urbano Braz, Jibin Zhang, Yang Liu, Peiheng Liu, Jiaxi Shen, Nanyumuzi Aori, Timothy A. Hacker, Henk Granzier, Wei Guo

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Figure 8

DCM-linked mutations in the RSRSP stretch contained within the D1 sequence element disrupt RBM20 nuclear localization.

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DCM-linked mutations in the RSRSP stretch contained within the D1 sequen...
(A) Schematic showing the domain structure of human RBM20 with the D1 sequence element shown. Critical RSRSP stretch is highlighted in red. Reported mutations in the RSRSP stretch in human RBM20 are listed along with the corresponding mutations in rat RBM20. Asterisks denote RBM20 mutants with localization data already available in the literature. (B) Representative images showing the localization of rat RBM20 harboring DCM-linked mutations corresponding those reported in the RSRSP stretch of human RBM20. Scale bars are 20 μm. All transfection experiments were repeated at least once for a total of 2 replicates. (C) Table displaying Grantham’s distances for DCM-linked amino acid mutations in the RSRSP stretch that disrupt RBM20 nuclear localization. Amino acid positions are given for rat RBM20. The number in parentheses corresponds to Grantham’s distance between the amino acid in the WT sequence and the mutant amino acid. Grantham’s distances were classified as conservative, moderately conservative, moderately radical, or radical as proposed by Li et al. (46). Asterisks denote RBM20 mutants with localization data already available in the literature.