@article{968a93f3e3e841fcb096609bc64735c5,
title = "RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis",
abstract = "Arginine–serine (RS) domain(s) in splicing factors are critical for protein–protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20S637A) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.",
keywords = "RNA-binding motif 20 (RBM20), cardiomyopathy, phosphorylation, pre-mRNA splicing, protein condensates, protein trafficking",
author = "Yanghai Zhang and Chunyan Wang and Mingming Sun and Yutong Jin and Braz, {Camila Urbano} and Hasan Khatib and Hacker, {Timothy A.} and Martin Liss and Michael Gotthardt and Henk Granzier and Ying Ge and Wei Guo",
note = "Funding Information: We would like to thank Dr. Donald L. Jarvis and Mrs. Melissa Ann Stuart at the University of Wyoming for their assistance in RBM20 expression in Sf2 cells, and to our lab manager Joan Parrish for her assistance for this project and the RNA Sequencing Core Facility at University of Wisconsin‐Madison for helping produce RNA‐seq data. We would like to thank the Gene‐Editing Core Facility for making the knock in mouse model at the University of Arizona. We would also like to thank the histology support from the Dairy Innovation Hub Histology Resource. Funding Information: This work was supported by the NIH NHLBI HL148733, NICHD HD101870, and NIGMS P20GM103432; the American Heart Association Foundation 16BGIA27790136 and 19TPA3480072; the Wisconsin Alumni Research Foundation (AAH4884); and the University of Wisconsin Foundation (AAH5964); YG would like to acknowledge NIH R01?grants, HL109810 and HL096971 and the high-end instrument grant S10OD018475 and HG would like to acknowledge R35HL144998 We would like to thank Dr. Donald L. Jarvis and Mrs. Melissa Ann Stuart at the University of Wyoming for their assistance in RBM20 expression in Sf2 cells, and to our lab manager Joan Parrish for her assistance for this project and the RNA Sequencing Core Facility at University of Wisconsin-Madison for helping produce RNA-seq data. We would like to thank the Gene-Editing Core Facility for making the knock in mouse model at the University of Arizona. We would also like to thank the histology support from the Dairy Innovation Hub Histology Resource. Funding Information: This work was supported by the NIH NHLBI HL148733, NICHD HD101870, and NIGMS P20GM103432; the American Heart Association Foundation 16BGIA27790136 and 19TPA3480072; the Wisconsin Alumni Research Foundation (AAH4884); and the University of Wisconsin Foundation (AAH5964); YG would like to acknowledge NIH R01 grants, HL109810 and HL096971 and the high‐end instrument grant S10OD018475 and HG would like to acknowledge R35HL144998 Publisher Copyright: {\textcopyright} 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.",
year = "2022",
month = may,
doi = "10.1096/fj.202101811RR",
language = "English (US)",
volume = "36",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "5",
}