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.

SR Protein Kinases Regulate the Splicing of Cardiomyopathy-Relevant Genes via Phosphorylation of the RSRSP Stretch in RBM20

“…Based on the data presented herein, it appears that increased phosphorylation of RBM20 through kinase overexpression does not promote nucleocytoplasmic transport of the protein as it does for the shuttling SR protein SRSF1 [ 53 ]. This finding is consistent with our recently published data showing that constitutive pseudo-phosphorylation of phosphorylation sites within the RS domain in RBM20 by Ser-to-Asp mutagenesis does not promote nucleocytoplasmic transport of the protein in H9c2 cells [ 26 ]. Nevertheless, a notable caveat is that there is evidence in the literature suggesting that SR protein shuttling is also dependent on RNA binding and HeLa and H9c2 cells to not express titin, the primary target of RBM20.…”

“…In vitro profiling of the kinase panel was performed at Reaction Biology Corporation, using the “HotSpot” assay platform. Briefly, 5 μM purified RBM20 protein [ 24 , 26 ] was incubated with 10 nM AKT2, 12.5 nM CLK1, 0.8 nM SRPK1, 1.5 nM CLK2, or 0.1 nM SRPK2 in reaction buffer (20 mM HEPES pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na 3 VO 4 , 2 mM DTT, and 1% DMSO). To assess the activity of different kinases, the same amount of AKT2, CLK1, SRPK1, CLK2, or SRPK2 was incubated with or without a standard substrate (crosstide for AKT2, MBP for CLK1 and CLK2, and RS peptide for SRPK1 and SRPK2) in the same reaction buffer.…”

“…Following in vitro kinase assays, RBM20 phosphorylation was assessed by using middle-down MS as previously described [ 26 ]. Briefly, RBM20 was digested and subjected to online liquid chromatography (LC)–MS and tandem MS (MS/MS) analyses [ 46 , 47 ], as well as offline high-resolution MS/MS analysis of phosphorylated peptides [ 48 ].…”

“…Genetic ablation of Rbm20 results in aberrant gene splicing and dilated cardiomyopathy (DCM) in rodents [ 2 , 16 ]. Furthermore, recent studies have shown that missense mutations in the arginine/serine (RS) domain—particularly within a conserved RSRSP stretch—of RBM20 also lead to aberrant splicing and severe DCM in patients and animal models [ 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 ].…”

“…An earlier study demonstrated that two Ser residues within the RSRSP stretch in RBM20 are constitutively phosphorylated [ 40 ]. Recently, our group confirmed phosphorylation of these residues (S638 and S640) and identified additional sites of phosphorylation in and outside the RBM20 RS domain by using middle-down mass spectrometry (MS) [ 26 ]. However, the kinases responsible for phosphorylating the RSRSP stretch in RBM20 remain to be identified.…”