Phosphorylation of the smooth muscle master splicing regulator RBPMS regulates its splicing activity

Barnhart, Michael D, Yang, Yi, Nakagaki-Silva, Erick E, Hammond, Thomas H, Pizzinga, Mariavittoria, Gooding, Clare, Stott, Katherine, Smith, Christopher WJ (November 2022) Phosphorylation of the smooth muscle master splicing regulator RBPMS regulates its splicing activity. Nucleic Acids Research, 50 (20). pp. 11895-11915. ISSN 0305-1048

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Abstract

We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.

Item Type:	Paper
Subjects:	bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification organs, tissues, organelles, cell types and functions bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression > phosphorylation bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression > post-translational modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > RNA binding protein bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA splicing organs, tissues, organelles, cell types and functions > tissues types and functions > smooth muscle organs, tissues, organelles, cell types and functions > tissues types and functions
CSHL Authors:	Nakagaki-Silva, Erick E.
Communities:	CSHL labs > Krainer lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	11 November 2022
Date Deposited:	09 Jan 2023 16:33
Last Modified:	11 Jan 2024 15:54
PMCID:	PMC9723635
URI:	https://repository.cshl.edu/id/eprint/40783

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