title: 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/).
authors: Department of Human Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors: Eleonora
authors: E
authors: de Klerk
authors: Department of Human Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors: Ivo F A C
authors: IF
authors: Fokkema
authors: Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, AMC/UvA, 1066CX 125 Amsterdam, The Netherlands.
authors: Klaske A M H
authors: KA
authors: Thiadens
authors: Biostatistics, Department for Health Evidence, Radboud University Medical Center, Postzone 133, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
authors: Jelle J
authors: JJ
authors: Goeman
authors: Center for Proteomics and Metabolomics, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
authors: Magnus
authors: M
authors: Palmblad
authors: Department of Human Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands.
authors: Johan T
authors: JT
authors: den Dunnen
authors: Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory, AMC/UvA, 1066CX 125 Amsterdam, The Netherlands.
authors: Marieke
authors: M
authors: von Lindern
authors: Department of Human Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands P.A.C._t_Hoen@lumc.nl.
authors: Peter A C
authors: PA
authors: 't Hoen
mesh_terms: D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D051379:Mice; D024510:Muscle Development / Q000235:genetics*; D032448:Myoblasts, Skeletal / Q000378:metabolism*; D010442:Peptide Chain Initiation, Translational; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods; D014158:Transcription, Genetic
chemical_list: D012333:RNA, Messenger; D012269:Ribosomal Proteins