We monitored translation at nucleotide resolution in a genome-wide high throughput manner, using ribosome profiling on the murine C2C12 cell line, a model for skeletal muscle differentiation. We simplified the existing protocol and developed a data analysis pipeline to characterize translation init

The formation of skeletal muscles is associated with drastic changes in protein requirements. These changes are safeguarded by tight control over transcription and mRNA processing. The importance of translational control is currently less clear. Regulation at translational level may affect both abundance and protein identity. Use of alternative translation initiation sites (TISs) in upstream open reading frames (uORFs) may increase or decrease protein synthesis, whereas alternative open reading frames (aORFs) produce different proteins. We applied ribosome profiling to monitor translation during myogenic differentiation of C2C12 cells. We simplified the existing protocol and developed a dedicated pipeline to identify TISs and quantify translation activity. We identified 5333 unannotated TISs, providing a catalogue of alternative TISs leading to uORFs and aORFs. By comparing ribosome profiling and DeepCAGE data, we found 312 genes that switched to an alternative TIS, not explained by alternative promoter usage. Many of these genes were ribosomal protein genes and genes involved in calcium signaling. The same pathways demonstrated discrepant total RNA and ribosome-associated RNA levels. Our data suggest that ribosome biogenesis and calcium signaling are most strongly regulated at the translational level, while the majority of differences in protein requirements during myogenesis are ensured by changes at transcriptional level.