Figure 5. Relative TE changes evoked by depleting 40S subunits are broadly comparable to those evoked by the tma∆∆ mutations or by increased eIF2α phosphorylation.

(A) Polysome profiles of WT strain H2994 and the rps26∆∆ mutant JVY09. Cells cultured in SC medium with 2% galactose and 2% raffinose instead of glucose at 30°C to log-phase were shifted to glucose-containing SC medium for 3 hr and treated with 50 μg/mL of CHX for 5 min before harvesting at 4°C. WCEs were resolved by sedimentation through sucrose gradients and scanned at 260 nm to visualize (from left to right) free 40S and 60S subunits, 80S monosomes, and polysomes. The mean polysome/monosome (P/M) ratios ± SEMs from five biological replicates are shown. (B) Volcano plot showing the log₂ ratios of relative TEs in the rps26∆∆ mutant versus WT cells (∆TE_rps26∆∆ values) for each mRNA (x-axis) versus negative log₁₀ of the FDR for the ∆TE_rps26∆∆ changes determined by DESeq2 analysis of ribosome profiling data for the 5187 mRNAs with evidence of translation (y-axis). The dotted line marks the 5% FDR threshold. mRNAs showing a significant increase (∆TE_rps26∆∆_up) or decrease (∆TE_rps26∆∆_down) in relative TE in the rps26∆∆ mutant versus WT cells at FDR < 0.05, are plotted in dark or light blue circles, respectively. (C) Cumulative distribution function (CDF) plots of log₂WT TE values for all mRNAs (black) and mRNAs exhibiting a significant increase (∆TE_rps26∆∆_up, solid dark blue) or decrease (∆TE_rps26∆∆_down, dotted light blue) in relative TE (at FDR < 0.05) in the rps26∆∆ mutant versus WT cells. p values were calculated using the Kolmogorov-Smirnov test. (D) Hierarchical clustering analysis of log₂TE changes observed for 248 mRNAs that exhibit significant TE changes in the tma∆∆ mutant versus WT cells, in SM-treated versus untreated WT cells, and also in the rps26∆∆ mutant versus WT cells, at p < 0.05 for all genotypes/conditions. The color scale for log₂ΔTE values ranges from 3.5 (dark blue) to −2.5 (dark red). (E–F) Notched box plots showing translation changes (log₂ΔTE) in the indicated mutant versus WT for mRNAs exhibiting a significant increase (∆TE_tma∆∆_up, N = 385, panel E) or decrease (∆TE_tma∆∆_down, N = 175, panel F) in relative TE in the tma∆∆ mutant versus WT cells at a 5% FDR threshold. A few outliers were omitted from the plots to expand the y-axis scale. p values were calculated using the Mann-Whitney U test for the differences between the indicated groups of mRNAs versus all mRNAs. (Columns 1–2 in panels (E–F) were previously compared in Figure 4—figure supplement 2A–B).

Figure 5—figure supplement 1. Reproducibility between biological replicates of ribosome footprint profiling and RNA-seq analyses for WT and the rps26∆∆ mutant.

(A–D) Scatterplots of normalized ribosome footprint (A, C) or mRNA (B, D) read densities for all expressed mRNAs for biological replicates of the WT (A, B) and the rps26∆∆ mutant (C, D). The densities were plotted from the reads mapped to the CDS of each gene per million mapped reads in the individual library of a biological replicate. Pearson’s correlation coefficient (r) for the plotted genes is indicated in each plot.

Figure 5—figure supplement 2. The rps26∆∆ mutations do not derepress GCN4 mRNA translation or confer 40S recycling defects.

(A) Results from ribosome profiling showing the normalized 80S ribosome reads from all mRNAs aligned with respect to their stop codons, for WT strain H2994 (light and dark blue) and the rps26∆∆ mutant JVY09 (yellow and orange), showing two replicates (a and b) for each strain. (B) Expanded view of normalized 80S reads from all mRNAs shown in (A) for the first 50 nt of the 3’UTRs. (C) Genome browser view of ribosome profiling data for GCN4 mRNA, showing RPF reads (Ribo) and mRNA reads (RNA) mapping across the transcription unit in WT, and in the rps26∆∆ mutant, showing two replicates (a and b) for each strain. The main CDS is shown schematically (orange). The calculated ∆TE_rps26∆∆ value is shown.

Figure 5—figure supplement 3. 40S subunit depletion in the rps26∆∆ mutant increases the relative TEs of mRNAs with attributes similar to mRNAs showing TE increases conferred by the tma∆∆ mutations.

(A) Frequency distribution plots of CDS length for all mRNAs (black) and mRNAs exhibiting a significant increase (∆TE_rps26∆∆_up, solid dark blue) or decrease (∆TE_rps26∆∆_down, dotted light blue) in relative TE (at FDR < 0.05) in the rps26∆∆ mutant versus WT cells. (B, D and E) Cumulative distribution function (CDF) plots of 5’UTR length (B), mRNA abundance in molecules per picogram of dry cellular weight (pgDW) (D), and mRNA half-life (E), for the groups of mRNA examined in (A). p values in panels A-B and D-E were calculated using the Kolmogorov-Smirnov test. (C) Notched box plots of context scores calculated for positions −3 to −1 and +4 of the main CDS AUGs for all mRNAs (white) and for the groups of mRNA, ∆TE_rps26∆∆_up (dark blue) and ∆TE_rps26∆∆_down (light blue), examined in (A). p values were calculated using the Mann-Whitney U test.

Figure 5—figure supplement 4. Effects of the rps26∆∆ mutations on the relative TEs of the "+Rps26" and "ΔRps26" groups of mRNAs.

Notched box plots showing the changes in relative TE observed in the rps26∆∆ mutant versus WT cells for all mRNAs and for the two groups of mRNAs found to bind preferentially to 40S subunits containing Rps26 ("+Rps26") or lacking Rps26 ("ΔRps26") by Ferretti et al., 2017. A few outliers were omitted from the plots to expand the y-axis scale. p values indicated in the panel were calculated using the Mann-Whitney U test.

Figure 5—figure supplement 5. Effects of rps29b∆ and rps0b∆ mutations on relative TEs of different groups of mRNAs calculated from the ribosome footprint profiling and RNA-seq data of Cheng et al., 2019.

(A–B) Notched box plots of relative TEs determined for single replicates of rps29b∆, rps0b∆, and two different WT strains for (A) five pentiles of 907–908 genes sorted according to increasing CDS length, and (B) all mRNAs versus 133 RPG mRNAs interrogated in the rps0b∆ strain and two different WT strains, or the 132 RPG mRNAs interrogated in the rps29b∆ strain.