Schematic models of the core of MAPK/ERK pathway and human eIF3 complex.

(A) The MAPK/ERK pathway activation by growth factors and receptor tyrosine kinases (RTKs). RTKs signal through GRB2-Sos and Ras to activate Raf-MEK-ERK signaling cascade. Active ERK phosphorylates numerous substrates, both nuclear and cytoplasmic, to affect gene transcription, protein translation, cell growth, proliferation and survival. Both RTKs and Ras also activate PI3K-AKT pathway that affect cell survival and growth. Conventional PAKs (PAK1-3) phosphorylate c-Raf and contribute to its activation. Created with Biorender.com. (B) eIF3 subunits forming the PCI/MPN octamer are indicated by the grey background. The rectangle marks the seven α-helices involved in formation of the 7-helix bundle. The Yeast-Like Core (YLC) comprising the eIF3 subunits a, b, g and i is depicted, and so is the eIF3-associated factor eIF3j. Arrows indicate subunits targeted by siRNA and subjected to Ribo-Seq in this study; eIF3 subcomplexes generated in individual knock-downs are boxed (adapted from (Wagner et al., 2016)).

DTEGs identified in eIF3dKD, eIF3eKD and eIF3hKD largely overlap.

(A) Principal component analysis of read count per gene of the RNA-Seq and Ribo-Seq libraries. (B - D) Volcano plots of significant (Padjusted < 0.05) DTEGs in individual knock-downs: eIF3dKD (B), eIF3eKD (C) and eIF3hKD (D). The volcano plots show the Log2 fold-change of TE (x-axis) versus the significance -Log10 p-adjusted value (y-axis). Genes without significant p-adjusted value are plotted in grey, downregulated DTEGs are plotted in blue and upregulated DTEGs in orange. For each plot, the top 10 most significant DTEGs are labeled with gene names. The Volcano plots were generated using modified script from Galaxy (Blankenberg et al, 2014). (E) Heatmap and dendrogram resulting from hierarchical clustering analysis of significant TE changes observed in eIF3dKD, eIF3eKD and eIF3hKD cells. (F - H) Venn diagrams depicting the overlap in all DTEGs (F), in upregulated DTEGs (G), and in downregulated DTEGs (H) among eIF3dKD, eIF3eKD and eIF3hKD. Based on the overlaps, DTEGs were divided in several subgroups for further analysis: eIF3dKD “all”, eIF3dKD “unique”, eIF3eKD “all”, eIF3eKD “unique”, eIF3dKD/eIF3eKD “common” and eIF3hKD “all”.

KEGG pathway enrichment analysis of the eIF3dKD- and eIF3eKD-associated upregulated DTEGs reveals upregulation of ribosomal proteins.

(A) Venn diagram of the 10 most significantly enriched KEGG pathways for eIF3dKD “all” and eIF3eKD “all” groups of upregulated DTEGs, highlighting that most of the pathways are common to both knock-downs. Lysosome and Protein processing in ER are highlighted in bold. The complete results of the KEGG enrichment analysis with corresponding p-values can be found in Supplementary Figure 5A. (B) Venn diagram as in A but displaying eIF3dKD “translation only” and eIF3eKD “translation only” groups of the upregulated DTEGs. (C) The bar chart shows the top 10 enriched KEGG terms for eIF3eKD “unique” upregulated DTEGs. Orange bars correspond to terms with significant p-values (< 0.05), grey bars correspond to terms with not significant p-values (> 0.05). The p-values were calculated by Enrichr gene set search engine. (D) The list of genes pre-selected for western blot analysis and a heatmap showing their respective log2 fold-change values from differential expression analysis of FP, TE and mRNA in eIF3eKD and eIF3dKD. Positive values indicating significant upregulation are in shades of orange. ns = not significant p-adjusted value. (E) Western blot analysis of selected ribosomal proteins performed in the indicated knock-downs and the NT control. GAPDH was used as a loading control. (F) Relative protein levels of selected ribosomal proteins normalized to GAPDH; NT control = 100%. Dots represent results from individual biological replicates. One sample t-test was used for statistical evaluation, P-values: * = P<0.05, ** = P<0.01, ns = not significant. (G) eIF3dKD, eIF3eKD and eIF3hKD do not influence the balanced ribosomal subunits production. The 60S/40S ratio was calculated from polysome profiles carried out in the presence of 50mM EDTA. Dots represent results from individual biological replicates. Paired t test was used for statistical evaluation, all downregulations were individually compared to NT. ns = not significant P-value. (H) Ribosomal content is increased in eIF3dKD and eIF3eKD. One representative polysome profile, carried out in the presence of 50mM EDTA, made from 10 million cells is shown in the upper panel. Relative ribosomal content normalized to NT control set to 100% is shown in the lower panel. Individual biological replicates are depicted as dots. One sample t-test was used for statistical evaluation, P-values: * = P<0.05, ** = P<0.01. All plots in (F-H) were created in GraphPad Prism version 8.4.3 for Windows.

Loss of eIF3 subunits leads to translational activation of mRNAs with 5’UTR TOP motifs.

(A-C) Scatterplots from translatome analysis of eIF3dKD (A), eIF3eKD (B), and eIF3hKD (C) with the location of transcripts harboring 5’ UTR TOP motifs (Philippe et al., 2020) colored (left panels). Middle and right panels show the empirical cumulative distribution functions of log2 fold changes in FP and total mRNA for the transcripts with TOP motifs. The background constituting all other transcripts are shown as grey curves. Significant differences between the distributions were identified using the Wilcoxon rank-sum test. Differences between the distributions at each quantile are indicated. Shift to the right indicates increased expression, while shift to the left indicates decreased expression. eIF3dKD and eIF3eKD show very significant increase of FPs of TOP mRNAs, suggesting mainly translational upregulation.

KEGG pathway enrichment analysis of the eIF3dKD- and eIF3eKD-associated downregulated DTEGs reveals downregulation of MAPK signaling pathways components.

(A) Venn diagram of the 10 most significantly enriched KEGG pathways for eIF3dKD “all” and eIF3eKD “all” groups of downregulated DTEGs, highlighting that most of the pathways are common to both knock-downs. The MAPK signaling pathway is highlighted in bold. The complete results of the KEGG enrichment analysis with corresponding p-values can be found in Supplementary Figure 7A. (B) The list of genes pre-selected for western blot analysis and a heatmap showing their respective log2 fold-change values from differential expression analysis of FP, TE and mRNA in eIF3dKD. Negative values indicating a significant downregulation are in shades of blue, while positive values showing significant upregulation are in shades of orange. ns = not significant p-adjusted value. (C) Western blot analysis of selected proteins constituting the MAPK/ERK pathway performed in the indicated knock-downs and the NT control. GAPDH was used as a loading control. (D) Relative protein levels of selected MAPK/ERK pathway proteins normalized to GAPDH; NT control = 100%. Dots represent results from individual biological replicates. One sample t-test was used for statistical evaluation, P-values: * = P<0.05, ** = P<0.01, *** = P<0.001, ns = not significant. (E) Western blot analysis of the phosphorylation status of the ERK1/2 proteins (left) and its relative quantification normalized to NT = 1 (right). One-sample t-test was used for statistical evaluation, **** = P <0.0001. (F) Western blot analysis of the phosphorylation status of the c-Jun transcription factor in cytoplasmatic and nuclear fractions in the indicated knock-downs and the NT control. The protein loading was 50ug of total protein for cytoplasmatic lysate and 30ug of total protein for nuclear lysate, as indicated. GAPDH was used as cytoplasmatic loading control and Lamin B1 was used as nuclear loading control. This experiment was repeated 3 times with similar results. All plots in (D-E) were created in GraphPad Prism version 8.4.3 for Windows.

KEGG pathway and GO enrichment analysis of the eIF3hKD-associated DTEGs reveals downregulation of proto-oncogene MDM2 and a defective stress-induced upregulation of ATF4.

(A) The bar chart shows the top 10 enriched KEGG terms for eIF3hKD “all downregulated” DTEGs. Blue bars correspond to terms with significant p-values (< 0.05), grey bars correspond to terms with not significant p-values (> 0.05). The p-values were calculated by Enrichr gene set search engine. (B) The bar chart shows the top 10 enriched GO Biological Process terms for eIF3hKD “all downregulated” DTEGs. Blue bars correspond to terms with significant p-values (< 0.05). The p-values were calculated by Enrichr gene set search engine. PCP; protein catabolic process. (C) Western blot analysis of the MDM2 expression preformed in the indicated knock-downs and NT control. GAPDH was used as a loading control; NT control = 100%. Dots represent results from individual biological replicates. One sample t-test was used for statistical evaluation, P-values: * = P<0.05, ** = P<0.01. Plots was created in GraphPad Prism version 8.4.3 for Windows. (D) Western blot analysis of the stress-induced upregulation of ATF4 expression performed in the indicated knock-downs and NT control. Before harvesting, cells were incubated for 3 hours with 1µM Thapsigargin to induce ER stress or with DMSO as a stress-free control. GAPDH was used as a loading control. (E) Normalized ribosomal footprint coverage along the ATF4 mRNA (first 450 nucleotides). Schematic with regulatory elements is shown at the bottom. Average of all three replicates is shown. Footprint coverage was normalized to all footprints mapping to the ATF4 mRNA. (F) Same as in (E) only for MDM2 mRNA.

Differential TE transcripts in eIF3dKD and eIF3eKD show negative correlation with the UTR length and uORF content while they positively correlate with GC content of their 3’ UTRs and coding sequences (CDS).

(A) Bar plot showing the Spearman correlation between the observed ΔTE values for all genes with assigned adjusted p-values in each knock-down and different mRNA features (5’ and 3’ UTR length or GC content, uORF content in 5’ UTR). *** = P < 10-20, ** = P < 10-10, * = P < 0.005. (B-C) Box and whisker plots comparing UTR lengths or GC content (in UTRs or CDS) among mRNAs with TE significantly increased (red, upregulated), decreased (blue, downregulated), or unchanged (white) in eIF3dKD (B) and eIF3eKD (C); *** = Padj < 10-10, ** = Padj < 10-5, * = Padj < 0.05, color indicates comparison set. (D) Same as in (B-C) but for the number of AUG-initiated uORFs in 5’ UTRs in the eIF3d, eIF3e and eIF3h knock-downs. Outliers are not shown for better clarity. (E) Histogram of the frequency of AUG-initiated uORFs per 5’ UTR in all transcripts listed in the uORFdb database that were assigned a p-value in this study (n=11450), for downregulated translation only DTEGs in eIF3dKD (n=1027) and for downregulated translation only DTEGs in eIF3eKD (n=618). (F) Venn diagram summarizing the main results of this study. Groups of significant DTEGs identified in given knock-downs (encircled – color-coded) are indicated by a blue arrow for downregulated or an orange arrow for upregulated. Results confirmed by western blotting are shown in bold. Ribosomal proteins were placed on the borderline between eIF3dKD and eIF3eKD because they were identified as eIF3eKD “unique upregulated” DTEGs, but their protein levels were elevated in both eIF3eKD and eIF3dKD, as shown by western blotting. The “MAPK/ERK signaling” is shown in bold italics because it is an independent phenomenon that could be detected only by western blotting.