Regulation of alternative splicing during myogenic differentiation of C2C12 cells

Post-transcriptional and co-transcriptional regulation of RNA processing play a crucial role in generating protein diversity, thus modulating many cellular processes such as cellular differentiation, reprogramming, and responses to external stimuli. RNA binding proteins participate to this fine regulation by binding to cis-regulatory elements (enhancers and silencers), helping the recruitment of the spliceosome and allowing alternative splicing (AS) of a multitude of exons. Genome-wide analyses have highlighted an extensive modulation of AS events during myogenic differentiation of C2C12 cells, suggesting a major role for AS in the acquisition of the muscle protein repertoire. Several splicing regulators undergo dynamic changes in nuclear abundance during myogenic differentiation of these cells. For instance, the polypirimidine tract binding protein (PTB) is downregulated while nPTB is upregulated. Moreover, we observed an increase of SAFB, EWS and SAM68 at the onset of myogenic differentiation. Strikingly, these RNA binding proteins are reported to interact with transcription factors and to play a role in co-transcriptional AS. For instance, SAM68 the Src-associated substrate belonging to a large class of RNA-binding proteins, has been shown to interact with androgen receptor (AR) and to play a role in the AS of AR target genes. Interestingly, AR expression is increased during proliferation and commitment of C2C12 cells and remains at high level in myotubes. Upon differentiation, AR is stored in the cytoplasm and it is activated after testosterone treatment. Our studies provide a new perspective of the functional coupling between transcription and alternative splicing at the onset of muscle differentiation through the interplay between splicing factors and master regulators of muscle differentiation, with the involvement of the AR.