Mass Spectrometry Analysis of RBM20 Phosphorylation and Its Role in Titin Splicing

Abstract

ObjectiveThe SR protein family has a characteristic domain which is rich in arginine and serine residues. They play significant roles in constitutive pre‐mRNA splicing and the phosphorylation status of them is important in the regulation of alternative splicing. RNA binding motif 20 (RBM20), a newly identified SR protein, mainly regulates splicing of a giant muscle gene titin, a major heart disease‐causing gene. However, the mechanism of RBM20 modulation on titin splicing is not well defined. We hypothesize that RBM20 phosphorylation plays an important role in titin splicing. In this study, we employed middle‐down mass spectrometry and in vitro splicing assay to explore the phosphorylation status of RBM20 and its role in titin splicing.MethodsBoth bacteria and insect cell expressed RBM20 were used for ProQ diamond staining and middle down mass spectrometry analysis. In vitro approaches with co‐transfection of mutated RBM20 and titin minigene, and a dual luciferase splice reporter assay were performed to explore the role of RBM20 phosphorylation in titin splicing.ResultsInsect cell expressed RBM20 was stained by ProQ diamond, a dye which is specifically used for phosphorylated protein staining in SDS‐PAGE gel, whereas ProQ diamond stained bands were not observed with bacteria expressed RBM20. Mass spectrometry analysis on the insect cell expressed RBM20 identified 16 phosphorylation sites with four of them located in RS domain. 16 mutated RBM20 plasmids on individual phosphorylation sites co‐transfected with titin minigene in HeLA cells, and the results indicated that only phosphorylation in RS domain affects titin splicing, and splice report assay further confirmed the results.ConclusionRBM20 is massively phosphorylated with 16 phosphorylation sites, and only RS domain phosphorylation plays a role in titin splicing. These results reveal that the phosphorylation sites on RS domain residues could be potential therapeutic targets for titin splicing‐induced heart disease.Support or Funding InformationNIFA‐USDA 1009266, NIH NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of WyomingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.