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. 2020 Aug 3;9:e58828. doi: 10.7554/eLife.58828

Figure 6. EDF1 recruits GIGYF2•EIF4E2 to collided ribosomes.

(A) (Left) UV (A258) absorbance across 10–50% sucrose gradients from lysates of HEK293 Flp-In TREx WT and ΔEDF1 cells left untreated (UT, blue trace) or treated with 1.8 µM emetine (EL, orange trace) for 15 min; fractions 6–8 pooled for light polysomes; fractions 9–11 pooled for heavy polysomes (n = 4). (Right) Schematic of polysome proteomics pipeline to monitor relative change in protein intensity in response to emetine treatment in light and heavy polysomes between WT and ΔEDF1 cells. (B, C) Volcano plot of log2 indicated ratio (x-axis) against -log10(p-value) (y-axis) for light (B) or heavy (B) polysomes. (D) UV (A258) absorbance across 10–50% sucrose gradients from lysates of HEK293-Flp-In TREx WT and ΔEDF1 cells left untreated (UT, blue trace) or treated with 1.8 µM emetine (EL, orange trace) for 15 min; TCA precipitated proteins from individual fractions were resolved by SDS-PAGE and analyzed by immunoblotting using GIGYF2 and uS3 antibodies. (n = 2) (E) Relative GFP intensity from HEK293 Flp-In TREx WT, ΔEDF1, ΔZNF598, and ΔEDF1ΔZNF598 cells transfected with the GFP-(KAAA)20-RFP stalling reporter without (white bars, non-targeting siRNA, SCRi) or with siRNA-mediated depletion of GIGYF2 and EIF4E2 (green bars; GIGYF2i•EIF4E2i). Error bars denote SD for n = 3. p-values were determined by one-way ANOVA and Tukey’s post hoc correction for multiple comparisons. See also Figure 6—figure supplement 1, Figure 6—figure supplement 2 and Figure 6—source data 1.

Figure 6—source data 1. Related to Figure 6B-C and Figure 6—figure supplement 1; Pooled sucrose gradient fractions (light and heavy polysomes) analysis with or without low-dose emetine treatment (1.8 µM, 15 min).
16plex TMT-MS3 analysis of HEK293 Flp-In T-REx cells (WT vs ΔEDF1).

Figure 6.

Figure 6—figure supplement 1. Identification of factors whose recruitment to collided ribosomes depends on EDF1.

Figure 6—figure supplement 1.

(A) Volcano plot of the fold change in the intensity of proteins identified in light polysomes of low-dose emetine-treated HEK293-Flp-In TREx WT cells compared to untreated cells (log2(EL/UT), x-axis) against -log10(p-value) (y-axis). (B) Volcano plot of log2(EL/UT) against -log10(p-value) for light polysomes in HEK293-Flp-In TREx ΔEDF1 cells. (C) Volcano plot of log2(EL/UT) against -log10(p-value) for heavy polysomes in HEK293-Flp-In TREx WT cells. (D) Volcano plots of log2(EL/UT) against -log10(p-value) for heavy polysomes in HEK293-Flp-In TREx ΔEDF1 cells.
Figure 6—figure supplement 2. Loss of EDF1 and GIGYF2 increases the bulk translational output of polyA- and Xbp1u-mediated stalling reporters.

Figure 6—figure supplement 2.

(A) Relative GFP intensity from HEK293-Flp-In TREx WT, GIGYF2i•EIF4E2i, ΔEDF1 and ΔEDF1 cell lines rescued with C-terminal Strep II-tagged full-length EDF1 (ΔEDF1:EDF1FL), or an N-terminal fragment of EDF1 (ΔEDF1:EDF1N-term, amino acids 1–74), or a C-terminal fragment of EDF1 (ΔEDF1:EDF1C-term, amino acids 73–148). Cells were transfected with the GFP-(KAAA)20-RFP stalling reporter. Error bars denote SD for n = 3. p-values were determined by one-way ANOVA and Tukey’s post hoc correction for multiple comparisons. (B) Whole cell extracts from ΔEDF1:EDF1FL, ΔEDF1:EDF1N-term and ΔEDF1:EDF1C-term cells were analyzed by SDS-PAGE and immunoblotted with a Strep II antibody. (C) 10–50% sucrose gradients from lysates of ΔEDF1:EDF1FL, ΔEDF1:EDF1N-term and ΔEDF1:EDF1C-term cells untreated (UT) or treated with low-dose emetine (EL, 1.8 µM, 15 min) were fractionated and analyzed by immunoblotting with a Strep II antibody. (D) Relative RFP:GFP ratio from HEK293 Flp-In TREx WT, ΔEDF1, ΔZNF598, and ΔEDF1ΔZNF598 cells transfected with the GFP-(KAAA)20-RFP stalling reporter without (white bars, non-targeting siRNA, SCRi) or with siRNA-mediated depletion of GIGYF2 and EIF4E2 (green bars; GIGYF2i•EIF4E2i). Error bars denote SD for n = 3. p-values were determined by one-way ANOVA and Tukey’s post hoc correction for multiple comparisons (related to Figure 6E). (E) The corresponding RFP:GFP ratios for the experiment described in A. Error bars, SD for n = 3. p-values were determined by one-way ANOVA and Tukey’s post hoc correction for multiple comparisons. (F) Relative GFP intensity from HEK293 Flp-In TREx WT, ΔEDF1, ΔZNF598, and ΔEDF1ΔZNF598 cells transfected with the GFP-RFP reporter with no intermediate stalling sequence [(KAAA)0 reporter] without (white bars, non-targeting siRNA, SCRi) or with siRNA-mediated depletion of GIGYF2 and EIF4E2 (green bars; GIGYF2i•EIF4E2i). Error bars represent SD for n = 3. p-values were determined by one-way ANOVA and Tukey’s post hoc correction for multiple comparisons. (G) The corresponding RFP:GFP ratios are depicted for the experiment described in F. (H) Relative Renilla luciferase (RLuc) intensity from HEK293 Flp-In TREx WT, ΔEDF1, ΔZNF598 cells, and ΔEDF1ΔZNF598 cells transfected with the RLuc-2A-XBP1u-2A-FLuc stalling reporter or a control reporter with no intervening XBP1u stalling sequence. Values for individual biological replicates (n = 5 or 6) plotted with median represented as black bar. p-values were determined by one-way ANOVA and Tukey’s post hoc correction for multiple comparisons. (I) The corresponding FLuc: RLuc ratios for the experiment described in H.