Assessing the translational landscape of myogenic differentiation by ribosome profiling.

The formation of skeletal muscles is associated with drastic changes in protein requirements known to be safeguarded by tight control of gene transcription and mRNA processing. The contribution of regulation of mRNA translation during myogenesis has not been studied so far. We monitored translation during myogenic differentiation of C2C12 myoblasts, using a simplified protocol for ribosome footprint profiling. Comparison of ribosome footprints to total RNA showed that gene expression is mostly regulated at the transcriptional level. However, a subset of transcripts, enriched for mRNAs encoding for ribosomal proteins, was regulated at the level of translation. Enrichment was also found for specific pathways known to regulate muscle biology. We developed a dedicated pipeline to identify translation initiation sites (TISs) and discovered 5333 unannotated TISs, providing a catalog of upstream and alternative open reading frames used during myogenesis. We identified 298 transcripts with a significant switch in TIS usage during myogenesis, which was not explained by alternative promoter usage, as profiled by DeepCAGE. Also these transcripts were enriched for ribosomal protein genes. This study demonstrates that differential mRNA translation controls protein expression of specific subsets of genes during myogenesis. Experimental protocols, analytical workflows, tools and data are available through public repositories (http://lumc.github.io/ribosome-profiling-analysis-framework/).

Selective mRNA translation in erythropoiesis.

The daily production of up to 1011 erythrocytes is tightly controlled to maintain the number of erythrocytes in peripheral blood between narrow boundaries. Availability of growth factors and nutrients, particularly iron, control the proliferation and survival of precursor cells partly through control of mRNA translation. General translation initiation mechanisms can selectively control translation of transcripts that carry specific structures in the UTRs. This selective mRNA translation is an important layer of gene expression regulation in erythropoiesis. Ribosome profiling is a recently developed high throughput sequencing technique for global mapping of translation initiation sites across the transcriptome. Here we describe what is known about control of mRNA translation in erythropoiesis and how ribosome profiling will help to further our knowledge. Ribosome footprinting will give insight in transcript-specific translation at codon resolution, which is of great value to understand many cellular processes during erythropoiesis. It will be of particular interest to understand responses to iron availability and reactive oxygen species (ROS), which affects translation initiation of transcripts harbouring upstream ORFs (uORF) and potential alternative downstream ORFs (aORF).