Identification and Validation of Notch2 as an Interacting Partner with the Cardiomyopathy Associated Protein RBM20

Abstract

Background: Mutations in the gene encoding the RNA-binding protein (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). RBM20 has been shown to regulate splicing of over 30 genes, most of which are implicated in ion homeostasis and muscle biology, but not much is known about its interacting partners. Using phage display profiling of the C-terminal fragment of recombinant RBM20, a group in Dr. Jaalouk’s lab identified a panel of potential interacting partners for RBM20, including Neurogenic Locus Notch Homolog Protein 2 (Notch2). Mutations in the latter are known to be implicated in the development of cardiomyopathies.

Our objective is to gain a better understanding of the molecular mechanisms by which mutations or down-regulation of the RBM20 gene lead to the development of the Dilated Cardiomyopathy disease (DCM). To this end, identifying the protein interacting partners of RBM20 protein may provide a better insight into the pathobiology of RBM20-mediated DCM.

Aims: In this study, we hypothesize that Notch2 protein interacts with RBM20 and that cardiomyopathies that arise due to mutations in RBM20 gene are partly mediated by a disruption in the interaction between RBM20 and Notch2 proteins. Thus, we first assessed the expression and the intracellular distribution of Notch2 protein and to determine if it co-localizes with RBM20. Second, we aimed to determine if there is direct interaction between RBM20 and Notch2 proteins.

Methods: For the purpose of studying the sub-cellular distribution and the co-localization between RBM20 and Notch2 proteins, we performed Immunofluorescence (IF) staining assays on Rhabdomyosarcoma (RD) cells. In addition, we performed Western Blotting followed by Co-Immunoprecipitation assays for the assessment of the direct protein-protein interaction between RBM20 and Notch2 proteins in RD cells. Results: Immunofluorescence staining results revealed an expression of RBM20 proteins in both the cytoplasm and the nucleus of 97.8 % of counted RD cells, yet these proteins were mostly distributed in the nucleus. Similarly, 59.5 % of RD cells expressed Notch2 proteins in both the cytoplasmic and nuclear compartments, yet most of these proteins were localized in the nucleus. In addition, Western Blotting experiments on RD cells showed an expression of Notch2 proteins having a molecular weight of ~ 106 KDa, and two RBM20 protein isoforms, one of ~ 125 KDa and the other of ~ 45 KDa. Finally, our Co-Immunoprecipitation assay suggested a protein-protein interaction between RBM20 and Notch2 proteins, where RBM20 was pulled down by Notch2 and appeared at ~ 129 KDa on the blot probed with RBM20 antibody.

Conclusion: Taken together, our data suggest a possible protein-protein interplay between RBM20 and Notch2 proteins. Additional experiments are needed to validate these findings and to decipher the importance of this interaction for the proper function of RBM20 protein.