Impact of uORFs in mediating regulation of translation in stress conditions

Fig. 1. Increased ribosome density in 5’UTR versus CDS in stress conditions is independent of uORFs. a Workflow of 5’UTR and CDS read mapping and quantification. We analyzed Ribo-Seq and RNA-Seq data from three experiments: #1 Scer.aa-, amino acid starvation in S. cerevisiae [27], #2 Scer. Oxi, oxidative stress in S. cerevisiae [21] and #3 Spom.N-, nitrogen starvation in S. pombe [16]. For each experiment we used two normal replicates and two stress replicates. We mapped the sequencing reads to 5’UTR and CDS separately, obtaining the corresponding CDS and 5’UTR tables of counts for each gene and sample. b Log10 ratio of 5’UTR to CDS Ribo-Seq reads in stress versus normal conditions in the three experiments. Each data point represents a gene. The number of Ribo-Seq reads is a proxy of ribosome density. A relative increase in the density of ribosomes in the 5’UTR in stress can be observed for the majority of genes in the three experiments. We discarded genes with less than 10 average mapped reads in both conditions. Tables with CDS and 5’UTR reads were merged to calculate the ratios. c Workflow of uORF read mapping and quantification. We defined uORFs in the 5’UTRs as all ATG to STOP…

Fig. 2. Changes in TE at the 5’UTR are not anti-correlated with changes in TE at the CDS. a Definition of changes in translational efficiency (TE). TE change is calculated as the log2TE between stress and normal conditions. b Correlation between log2TE in 5’UTR and CDS. We discarded genes with less than 10 mapped reads in both conditions, taking the average between replicates, in Ribo-Seq and/or RNA-Seq experiments Number of datapoints: Scer.aa- 438; Scer. Oxi 1503; Spom.N- 2039. Spearman correlation: Scer.aa- 0.046; Scer. Oxi 0.19; Spom.N- 0.27

Fig. 3. Identification of genes regulated at the transcriptional and translational levels during stress. a Workflow describing differential gene expression (DGE) and translational efficiency (TE) analyses using Ribo-Seq and RNA-Seq reads. In each experiment we subsampled the original table of counts as to have the same total number of reads in each Ribo-Seq and RNA-Seq sample considered. This ensured the results would not be biased by lack of statistical power in the samples with less coverage. The data was used to define regulatory classes for different sets of genes. b Correlation between replicates and between RNA-Seq and Ribo-Seq samples. Two representative examples are shown, data is counts per million (CPM). c Definition of regulatory classes after DGE analyses. Transcriptional change: Genes that showed significant up-regulation or down-regulation using both RNA-Seq and Ribo-Seq data. Translational change: Genes that showed significant up-regulation or down-regulation only with Ribo-Seq data. Post-transcriptional buffering: Genes that showed significant up-regulation or down-regulation only with RNA-Seq data. The axes represent logFC between stress and normal conditions. d Fraction…

Fig. 4. Examples of uORF-mediated translational regulation. GCN4: well-studied case in which high translation of uORFs that are proximal to the CDS has an inhibitory effect on the translation of the main protein in normal conditions. Decreased rate of translation of these uORF during stress allows the protein to be expressed. The plot shows a profile of Ribo-Seq reads in normal and stress conditions, Scer. Oxi dataset. FC CDS stress vs normal RNA-Seq: 0.84; FC CDS stress vs normal Ribo-Seq: 3.51. fi1: functional analog to GCN4 in S. pombe. The Fil1 mRNA shows changes in ribosome density in several uORFs, suggesting that it could also be regulated by uORFs. Data shown is from the Spom.N- experiment. FC CDS stress vs normal RNA-Seq: 1.01; FC CDS stress vs normal Ribo-Seq: 3.02. ubi4: the S. cerevisiae polyubiquitin gene Ubi4 has been involved in the response to different types of stress. Here we could observe that the translation of Ubi4 was strongly activated during nitrogen depletion in S. pombe and that this was accompanied by a strong decrease in the translation of two uORFs in the 5’UTR (decrease in the number of Ribo-Seq mapped reads 10–20 fold). FC CDS stress vs normal RNA-Seq:…