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 6

Disruption of the D1 core NLS sequence element abolishes RBM20 nuclear localization.

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Disruption of the D1 core NLS sequence element abolishes RBM20 nuclear l...
(A) In silico identification of SRSF1 core NLS-like sequence elements in the RS domain of rat RBM20. Conserved amino acids are shown in green and blue denotes a positively charged amino acid. (B) Representative ICC images showing rat RBM20 deletion construct localization in transfected H9c2 cells. (C) Schematic showing rat RBM20 Ala substitution constructs for in vitro expression in H9c2 cells. (D) Representative ICC images showing rat RBM20 Ala substitution construct localization in transfected H9c2 cells. Scale bars are 20 μm. (E) Schematic showing human RBM20 domain structure with a zoom-in on the RS domain sequence and DCM-associated mutations listed. The human-specific segment in the D2 sequence element is highlighted in light blue. (F) Representative ICC images showing the localization of human RBM20 constructs harboring DCM-associated mutations in the D1 and D2 sequence elements. Scale bars are 20 μm. All transfection experiments were repeated at least once for a total of 2 replicates.