Effects of siRNA depletion on gene expression during C2C12 skeletal muscle differentiation initiation
In humans, skeletal muscles comprise nearly 40% of total body mass, which is maintained throughout adulthood by a balance of muscle protein synthesis and breakdown. Cellular amino acid (AA) levels are critical for these processes, and mammalian cells contain transporter proteins that import AAs to maintain homeostasis. Until recently, the control of transporter regulation has largely been studied at the transcriptional and post-translational levels; however, our recent work demonstrates that the RNA-binding protein YBX3 is critical to intracellular sustain AA in human cells. Here we report that YBX3 post-transcriptionally controls the levels of specific AA transporter messenger (m)RNAs in skeletal muscle cells, which impacts the intracellular levels transported by these proteins. Further, we find that reduction of YBX3 protein reduces proliferation during skeletal muscle differentiation, and that YBX3 expression increases substantially during skeletal muscle differentiation, which is independent to changes in itsmRNA levels. Taken together, our findings suggest that YBX3 regulates AA transport in skeletal muscle cells and that its expression is critical to maintain skeletal muscle cell proliferation during differentiation. Overall design: To identify genes whose expression is controlled by YBX3, we used high-throughput RNA sequencing (RNAseq) in C2C12 mouse skeletal muscle cells depleted of YBX3 by small interfering RNA (siRNA).  We assessed changes in transcripts levels in myoblasts before (proliferating) and one day after differentiation initiation (differentiating), which allowed for cells to be harvested concurrently at 48 hours post siRNA transfection.