Assessing the translational landscape of myogenic differentiation by ribosome profiling

Abstract

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/).

Translation of CircRNAs

“…We utilized a Ribo-seq dataset from rat brain and liver, and one from mouse C2C12 cells (de Klerk et al., 2015, Ori et al., 2015). We found unequivocal RFP reads for 34 and 158 circRNAs in the rat and mouse samples, respectively (Tables S5 and S6).…”

Decoding sORF translation – from small proteins to gene regulation

“…Lack of this 5'-most exon in differentiated myotubes could contribute to the increased Cryab protein production. 120,121 Because alternative TSS usage is common during vertebrate embryogenesis 122,123 and in different cell types, [124][125][126][127] such a transcription-based control of uORF regulation could have broad implications in developmental regulation of gene expression.…”

Extensive translational regulation during seed germination revealed by polysomal profiling

“…Polysome occupancy is partly synonymous with the term polysome loading and is used as a proxy of translational efficiency in the literature. This system has been successfully applied to investigate translational control in, for example, Saccharomyces cerevisiae (Arava et al, 2003;Halbeisen & Gerber, 2009;Ingolia et al, 2009), Aspergillus fumigatus (Krishnan et al, 2014), a mammalian cell line (de Klerk et al, 2015) and Arabidopsis thaliana (Jiao & Meyerowitz, 2010;Liu et al, 2012Liu et al, , 2013Juntawong et al, 2014;Basbouss-Serhal et al, 2015).…”