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

Abstract

(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy

“…Notably, all but three of the SRPK3/TTN families did not present cardiac involvement. Speculatively, abnormal RBM20 phosphorylation by SRPK3 in the heart would be overcome by ubiquitously expressed kinases, as shown recently for cdc2-like kinases (CLKs) and protein kinase B (AKTs) 25 and supported by our in vitro phosphorylation assay. Although RBM20 has not yet been associated with skeletal muscle disease, it has been shown to be DE across different skeletal muscles, where it regulates Z-band and M-band TTN splicing 48 .…”

“…We hypothesized that RBM20 might be a phosphorylation substrate of SRPK3 and the mediating link between SRPK3 and titin, as previously shown for SRPK1 (ref. 25 ). To investigate this, we cotransfected an RBM20 reporter (RBM20 517–664 -V5) into 293T cells with or without a GFP-SRPK3 construct.…”

Mislocalization of pathogenic RBM20 variants in dilated cardiomyopathy is caused by loss-of-interaction with Transportin-3

“…In addition to TNPO3 , we selected other hits with less significant FDR scores ( CLDN14 , GALE , ADAMTS16 , SLC29A2 , CEBPB , UBQLNL , TRIM33 , PMM2 , TRIM24 , IPPK , XPO6 , Supplementary Fig. 5e ) and based on prior knowledge about their relevance for RBM20 function 18 , 36 , 37 ( TTN , AKT2 , SPRK1 , CLK1 , LMNA ). We tested these potential positive hits by constructing single-gene KOs in HeLa cells and assessing their impact on RBM20-WT localization by ICS and fluorescence microscopy.…”

“…It has been observed that the binding of TNPO3 to its cargo can be phosphorylation-dependent 40 – 42 or independent 46 . In the case of RBM20, serine residues in the RS domain are normally phosphorylated 26 , 35 , 37 , 44 , however, phosphomimetic amino acid substiutions do not rescue the localization phenotype 26 . Importantly, both pooled and individual CRISPR KOs of kinases AKT2 , CLK1 , and SPRK1 —previously shown to phosphorylate RBM20 37 —did not impact RBM20 localization (Fig.…”

Mapping alternative splicing events in colorectal cancer

“…Recent studies have increasingly focused on the role of post-translational modifications, especially phosphorylation, in AS regulation [ 41 – 43 ]. For example, AMP-activated protein kinase (AMPK) and SR protein kinase 1 (SRPK1) phosphorylate SRSF1 [ 44 , 45 ]; PPM1G phosphorylates SRSF3 [ 46 ]; CDK11 phosphorylates SF3B1 [ 47 ]; oncogenic KRAS regulates SFs phosphorylation [ 48 ]; SR protein kinase regulates the phosphorylation of the RSRSP stretch in RBM20 [ 49 ]; ERK2 regulates the phosphorylation of Thr113 and Thr118 [ 50 ]; and the C-terminal domain of RNA polymerase II (Pol II CTD) is also phosphorylated [ 51 ]. These phosphorylation events in SFs and RBPs have various regulatory effects on AS.…”