Alternative Splicing of Nonmuscle Myosin Heavy Chains

National Heart, Lung, And Blood Institute

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Abstract

Myogenesis is regulated by the coordinated expression of muscle regulatory factors that includes Mbln1, Ptbp1, Cugbp1, Cugbp2 and Rbfox2. Rbfox2 is a UGCAUG sequence-specific RNA binding protein and plays an important role in regulating alternative splicing transitions during skeletal muscle differentiation. We and others have previously reported that the Rbfox2 gene undergoes tissue-specific alternative splicing and produce multiple isoforms specific to brain, heart and skeletal muscle. However, the function of Rbfox2 isoforms and their associated protein interaction and signaling pathways in skeletal muscle differentiation are largely unknown. Thus, we used a live cell proximity-labeling proteomics approach (BioID) to identify a spectrum of new protein interactions to Rbfox2 in C2C12 myoblast cells. This technique is particularly useful in identifying weak or transient interactions in living cells that are not detected by co-immunoprecipitation or yeast two-hybrid systems. In this study, we used three different Rbfox2 isoforms either containing ubiquitously expressed (B40) or a muscle specific (M43) exon or lacking an RNA recognition motif. The BioID fusion Rbfox2 isoforms are predominantly localized to the nucleus of the cell, and regulates the alternative splicing of an exogenously expressed Non-muscle Myosin Heavy Chain IIB mini-gene, reflecting native Rbfox2 function and subcellular distribution. To identify Rbfox2-proximal proteins in living cells, biotin was added to the cells expressing myc-BirA*Rbfox2 or myc-BirA*control followed by streptavidin pull-down and mass spectrometry analysis. Following mass spectrometry analysis, we filtered the data for a minimum fold change of >3 over control. This analysis identified 453 proteins proximal to at least one Rbfox2 isoform and also identified known interactors of Rbfox2 such as Ataxin2. Of the 453 total proteins identified, 94 were common to all three Rbfox2 isoforms. This common set of 94 proteins was highly enriched for gene ontology terms related to the spliceosomal complex assembly, N6-methyladenosine-containing RNA binding and histone deacetylase binding that suggests a novel physiological role for Rbfox2.