Assessing the translational landscape of myogenic differentiation by ribosome profiling.
Bottom Line: 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.Enrichment was also found for specific pathways known to regulate muscle biology.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.
Affiliation: Department of Human Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands.Show MeSH
Mentions: To distinguish between uORFs and alternative extended N-termini, we focused on TISs located in the annotated 5′-UTRs, and we classified them based on their reading frame in relation to the pORF and the presence of stop codons. Sixty percent of the detected TISs located in the 5′-UTRs were leading to stop codons prior the start of the pORFs (corresponding to 1274 TISs and 380 TISs in myoblasts and myotubes, respectively) (Figure 6A). The length of these uORFs ranged from 1 to more 100 amino acids (Figure 6C), but the majority (∼85%) were between 1–30 amino acids (50% was shorter than 10 amino acids). The remaining 40% of the TISs located in 5′-UTRs were not leading to stop codons prior the start of the pORF, but ∼72% of these uORFs was in a different reading frame than the pORFs, leading to overlapping uORFs, whereas the remaining 28% was in-frame with the pORF, suggesting the presence of isoforms with extended N-termini (Figure 6B). The length of the overlapping uORFs was longer than the one of the non-overlapping uORFs, reaching up to 400 amino acids and with only ∼40% being shorter than 30 amino acids (Figure 6D). We then investigated whether the usage of TISs in the 5′-UTRs sequences was associated with the presence of known regulatory elements, such as Internal Ribosome Entry Sites (IRESs) and Terminal Oligopyrimidine Tracts (5′ TOP). A significant enrichment of predicted IRES was found in transcripts with TISs in the 5′-UTRs, compared to transcripts for which we detected TISs only in the annotated start codon and in the coding region. 36% of the transcripts containing TISs in the 5′-UTR had IRESs (Supplementary Table S17), whereas the percentage dropped to 24 for transcript without TISs in their 5′-UTRs in myoblasts (27 against 20% in myotubes, respectively). No significant enrichment was found for predicted 5′ TOPs (Supplementary Table S18), and overall the percentage of transcripts with a TIS in the 5′-UTR and the presence of a predicted 5′ TOP was lower compared to the percentage of transcripts containing predicted IRES (∼4% for both myoblasts and myotubes). These results suggest that for these genes uORFs do not play an important role in the regulation of mRNAs starting with a 5′ TOP in myogenesis, whereas they may favor the use of IRESs in a subset of genes.
Affiliation: Department of Human Genetics, Leiden University Medical Center, Postzone S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands.