Functional characterization of Rbfox mediated protein interaction in skeletal muscle differentiation
Wednesday, September 13, 2017 — Poster Session I
- T Govindarajan
- RS Adelstein
- S Kawamoto
Rbfox1 and Rbfox2, sequence-specific RNA binding proteins, regulate alternative splicing of a variety of transcripts essential for cardiac and skeletal muscle development. Our lab has previously reported that both Rbfox1/2 undergo tissue-specific alternative splicing and produce multiple isoforms specific to brain, heart and skeletal muscle. However, the functional role of Rbfox1/2 isoform regulation and associated protein interaction remains to be elucidated. Here, we used a proximity-labeling proteomics approach called BioID (Biotin identification) to identify the protein interaction network of individual Rbfox1/2 isoforms. We started our experiments using the C2C12 mouse myoblast cell line as a model since C2C12 cells can be differentiated into myotubes in culture, similarly to primary myoblasts. We first used immunofluorescence staining to compare the subcellular localizations of the BioID-Rbfox1/2 with the native protein. The results demonstrate that the fusion protein’s subcellular localization is not altered in comparison to native Rbfox1/2 isoforms. Additionally, all the BioID-Rbfox1/2 isoforms with the RNA recognition motif exhibited similar splicing activities as native proteins. Next, we tested the biotinylation of endogenous proteins in C2C12 cells expressing BioID-Rbfox1/2 isoforms, either in the presence or absence of exogenous biotin, using Western blots probed with Streptavidin. The results indicate that in the presence of exogenously added biotin, BioID-Rbfox1/2 isoforms strongly stimulate biotinylation of a wide range of endogenous proteins. In future, we plan to use affinity capture the biotinylated proteins in C2C12 cells expressing individual BioID-Rbfox1/2 isoforms and identify them by mass spectrometry for functional characterization.
Category: Cell Biology