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. 2021 Apr 26;10:e66904. doi: 10.7554/eLife.66904

Figure 5. Pelo/Hbs1l deficiency alters translation regulation and reprograms the translatome.

(A) Translational efficiency (TE) of genes that are transcriptionally upregulated or downregulated was compared to the remaining (‘other’) genes from Pelo-/- (blue) and Hbs1l-/- (red) MEFs, respectively. Downward direction of arrows indicates significant decrease in translational efficiency of transcriptionally upregulated genes in Pelo-/- and Hbs1l-/- MEFs. Upward direction of arrows indicates significant increase in translational efficiency of transcriptionally downregulated genes in Pelo-/- and Hbs1l-/- MEFs. (B) KEGG pathway analysis of genes from Pelo-/- MEFs in which the translational efficiency (TE Pelo-/-, adj. p≤0.05) is opposite to their transcriptional expression (DE mRNA Pelo-/-, q-value ≤0.05). Italicized pathways indicate pathways that overlapped with enriched pathways of differentially translated genes in Pelo-/- MEFs (DE RPF Pelo-/-, adj. p≤0.05) (Figure 5—figure supplement 1E). The red dashed line indicates the significance threshold (p=0.05). (C) Ingenuity pathway analysis (IPA) of genes with differential translational efficiency from Pelo-/- (TE Pelo-/-) and Hbs1l-/- (TE Hbs1l-/-) MEFs. EIF2 and mTOR/p70S6K signaling are in italics. The red dashed line indicates the significance threshold (p=0.05). (D) Western blot analysis of p-eIF2αS51 and p-p70S6KT389 using lysates of tamoxifen-treated control (Pelofl/fl) and Pelo-/- MEFs at Day 2 (Passage P1). Vinculin was used as a loading control. (E) Levels of p-eIF2αS51 or p-p70S6KT389 were normalized to total level of eIF2α or p70S6K, and phosphorylation levels are relative to those of control (Pelofl/fl). Data represent mean + SD. (F) Translational efficiency (TE) of genes with translational regulation by mTOR via their 5’TOP motif was compared to the remaining (‘other’) genes from Pelo-/- (blue) and Hbs1l-/- (red) MEFs. Upward direction of arrows indicates significant increase in translational efficiency of 5’TOP genes. 4-OHT, 4-hydroxytamoxifin; 5’TOP, 5’terminal oligopyrimidine motif. Student’s t-test (E); Wilcoxon rank-sum test was used to determine statistical significance (A, F). ns, not significant; **p≤0.01.

Figure 5—source data 1. Pelo/Hbs1l deficiency alters translation regulation and reprograms the translatome.

Figure 5.

Figure 5—figure supplement 1. Pelo/Hbs1l deficiency alters translational gene expression of multiple pathways.

Figure 5—figure supplement 1.

(A) Translational efficiency (TE) of Pelo-/-- and Hbs1l-/--pausing genes was compared to the TE of the remaining (‘other’) genes from Pelo-/- (blue) and Hbs1l-/- (red) MEFs. Upward direction of arrows indicates significant increase in translational efficiency of Pelo-/-- and Hbs1l-/--pausing genes. (B) Analysis of differentially translated genes in Pelo-/- (DE RPF Pelo-/-, adj. p≤0.05, left) and Hbs1l-/- (DE RPF Hbs1l-/-, adj. p≤0.05, right) MEFs are shown in gray. Pelo-/-- and Hbs1l-/--specific pausing genes (z-score ≥10, detected in all three biological replicates) that are differentially translated are highlighted. Twofold changes in expression are indicated by the black dashed lines. (C) Differentially translated genes in Hbs1l-/- MEFs (DE RPF Hbs1l-/-, adj. p≤0.05, x-axis, red) were plotted against genes that are differentially translated in Pelo-/- MEFs (DE RPF Pelo-/-, adj. p≤0.05, y-axis, blue). Genes those translation was significantly different in both Hbs1l-/- and Pelo-/- MEFs are shown in purple. (D) Differential translation of genes in Pelo (DE RPF Pelo-/-, adj. p≤0.05) and Hbs1l (DE RPF Hbs1l-/-, adj. p≤0.05) was compared to that of Pelo-/-- and Hbs1l-/--specific pausing genes. Downward direction of arrows indicates significantly reduced changes in RPFs from the pausing genes compared to non-pausing (‘other’) genes. (E) KEGG pathway analysis of differentially translated (up- and downregulated) genes (DE RPF Pelo-/-, adj. p≤0.05) in Pelo-/- MEFs. Top nine of the significantly enriched pathways are shown. The red dashed line indicates the significance threshold (p=0.05). Pathways that are positively (ribosome, ribosome biogenesis) and negatively (lysosome, autophagy) regulated by mTORC1 are in italics. (F) Ingenuity pathway analysis (IPA) of differentially transcribed genes in Pelo (DE mRNA Pelo-/-) and Hbs1l (DE mRNA Hbs1l-/-). EIF2 and mTOR/p70S6K signaling are in italics. The red dashed line indicates the significance threshold (p=0.05). RPF, ribosome protected fragments. Wilcoxon rank-sum test was used to determine statistical significance (A, D); Pearson coefficient (r) was determined to analyze linearity of gene expression changes (C).