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. 2021 Dec 13;10:e69729. doi: 10.7554/eLife.69729

Figure 6. Genetic inactivation of Eef2k restores translation rates following Rpl24Bst mutation.

(A) 35S-methionine incorporation to determine relative protein synthesis by in Apcfl/fl KrasG12D/+ small intestinal organoids wild-type or mutant for Rpl24 or with both Rpl24 andConsistent with this, the Eef2k mutations. Data are represented ± standard error of the mean (standard error of the mean, SEM) with significance determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison. N = 3 per genotype, each representing an independent organoid line. (B) Ribosome run-off rate determined in Apcfl/fl KrasG12D/+ small intestinal organoids mutant or wild-type for Rpl24 or with both Rpl24 and Eef2k mutations. Data are the mean of three biologically independent organoid lines represented ± SEM with significance determined by Mann–Whitney U test. Raw data are available in Figure 6—figure supplement 1A. The run-off rate for Apcfl/fl KrasG12D/+ control organoids is reproduced from Figure 3E. (C) Schematic representation of findings in Apc-deficient Kras-mutant mouse and organoid models. Top: RPL24 expression maintains translation and proliferation by suppressing the phosphorylation of eEF2 by limiting eEF2K activity. Middle: reduced expression of RPL24 activates eEF2K, increasing P-eEF2, reducing translation elongation and suppressing tumorigenesis and proliferation. Bottom: inactivation of eEF2K reverts the phenotype in Rpl24Bst cells, due to the inability to phosphorylate and suppress eEF2. Elevated elongation rates correlate with increased proliferation following inactivation of eEF2K.

Figure 6.

Figure 6—figure supplement 1. Inactivation of Eef2k restores translation elongation speed in Rpl24Bst mutant mice.

Figure 6—figure supplement 1.

(A) Polysome profiles from sucrose density gradients of Apcfl/fl KrasG12D/+ Rpl24Bst/+ or Apcfl/fl KrasG12D/+ Rpl24Bst/+ Eef2kD273A/D273A small intestinal organoid cultures, pre-treated with harringtonine for 5 min/300 s (H300) or untreated (H0). These traces are representative of those analysed for the run-off rates shown in Figure 6B. (B) Representative sucrose density profiles generated from Apcfl/fl KrasG12D/+ intestinal extracts with or without the Rpl24Bst mutation and inactivation of eEF2K. Subpolysomal components and polysomes are labelled, and the polysomes have been split pictorially into light and heavy. To the right of this is quantification of the heavy:light polysome ratio. Data show the mean ± standard error of the mean (standard error of the mean, SEM) of 5, 3, and 3 mice reading from left to right. Significance was determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison.
Figure 6—figure supplement 2. Rpl24Bst mutation has no effect on P-ERK or P-ACC.

Figure 6—figure supplement 2.

(A) Immunohistochemistry (IHC) staining for P-ERK T202/T204 in intestinal tissue form Apcfl/fl KrasG12D/+ or Apcfl/fl KrasG12D/+ Rpl24Bst/+ mice. Representative images from each genotype are shown on the right and H-score quantification from three animals per genotype on the left. (B) Western blotting on lysates from Apcfl/fl KrasG12D/+ or Apcfl/fl KrasG12D/+ Rpl24Bst/+ organoids, for P-ACC S79, ACC, and β-actin as a sample control. All scale bars are 50 μm.
Figure 6—figure supplement 2—source data 1. Left: data from Figure 6—figure supplement 2B.
Right: original western blotting images with areas for each antibody annotated and boxed in red.