Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development

Abstract

To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly(717)Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly(717)-to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1(-/-) mice, eEF2(G717R/G717R) MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2(G717R/G717R) mice showed milder phenotypes than OVCA1(-/-) mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2(G717R/G717R)/OVCA1(-/-) double mutant mice displayed the milder phenotypes of the eEF2(G717R/G717R) mice, suggesting that the embryonic lethality of OVCA1(-/-) mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent -1 frameshifting during translation and show that the Gly(717)-to-Arg mutation cannot rescue this defect.

Fig. 1.

The eEF2^G717R/G717R mice are delayed in development. (A) The eEF2 G717R point mutation introduced into the mouse eEF2 locus. The mutation also generates a SacII site to facilitate genotyping as shown in D. Diphthamide is assembled on residue H⁷¹⁵. (B) Gene targeting of eEF2 by homologous recombination using the gene targeting vector containing the eEF2 G717R mutation. The Neo cassette flanked by two loxP sites was removed by breeding the resulting targeted mice with Cre general deleter mice. (C) Representative Southern blot analyses of offspring of eEF2-targeted mice. Genomic DNA digested with EcoRV was hybridized with a 3′ external probe. (D) Representative PCR genotyping analyses. A DNA fragment spanning the mutation site (with SacII site introduced, A) was amplified and digested by SacII, and separated on agarose gel. Note that the mutated fragment was digested into two smaller fragments, whereas the WT fragment remained intact. (E and F) Embryos at E14.5 (E) and E17.5 (F) with various genotypes. Note that eEF2^G717R/G717R embryos at E14.5 are similar in size to WT and eEF2^+/G717R embryos but significantly smaller at E17.5. (G) ADP-ribosylation analyses of eEF2^+/+, eEF2^+/G717R, and eEF2^G717R/G717R embryos. Tissue lysates from eEF2^+/+, eEF2^+/G717R, and eEF2^G717R/G717R embryos were incubated with DT for 30 min in the presence of biotin-NAD. The transfer of biotin-ADP ribose to eEF2 was detected by Western blotting using a streptavidin conjugate. The relative amounts of the eEF2 species that could be ADP ribosylated (after normalization to total eEF2) from eEF2^+/+ and eEF2^+/G717R tissues are indicated under the lanes. (H) Body weight trends of male eEF2^+/+ (n = 5 ∼ 12 for each time point), eEF2^+/G717R (n = 5 ∼ 16 for each time point), and eEF2^G717R/G717R (n = 4) mice.

Fig. 2.

ADP ribosylation and reactivity of various species of eEF2 to anti-eEF2 carboxyl terminus antibody (A and B). Cell lysates from OVCA1^+/+, OVCA1^+/−, and OVCA1^−/− MEFs (A) and from eEF2^+/+, eEF2^+/G717R, and eEF2^G717R/G717R MEFs (B) that had been incubated with or without PA and FP59 (1,000 ng/mL and 500 ng/mL, respectively) for 2 h were separated by native PAGE or SDS/PAGE and analyzed by Western blotting using an anti-eEF2 carboxyl terminus antibody (sc-25634; Santa Cruz Biotechnology).

Fig. 3.

The eEF2 G717R but not the eEF2 from OVCA1^−/− cells retains full activity in polypeptide elongation (A and B). MEF protein synthesis analyses. MEFs with eEF2^+/+, eEF2^+/G717R, or eEF2^G717R/G717R genotypes (A) and with OVCA1^+/+, OVCA1^+/−, or OVCA1^−/− genotypes (B) incubated with or without PA and FP59 (1,000 ng/mL and 500 ng/mL, respectively) for 2 h were incubated with [H³]-leucine–containing medium for various times as indicated. The incorporated [H³]-leucine was quantitated by a β-scintillation counter as scintillation cpm. (C and D) MEF growth rate analyses. MEFs with eEF2^+/+, eEF2^+/G717R, or eEF2^G717R/G717R genotypes (in C, seeded at 1 × 10⁵ cells per well in 6-well plates) and MEFs with OVCA1^+/+, OVCA1^+/−, or OVCA1^−/− genotypes (in D, seeded at 1.5 × 10⁵ cells per well) were allowed to grow and were counted at times as indicated.

Fig. 4.

The lack of diphthamide increases the rate of −1 frameshifting in protein synthesis (A). Schematic representation of the −1 frameshifting reporter system. pDual-HIV(−1) encodes a fusion protein of renilla luciferase and firefly luciferase with firefly luciferase in −1 frame. Thus, a fusion protein containing the firefly luciferase is translated only when a −1 frameshift occurs. (B) MEFs isolated from eEF2^+/+, eEF2^G717R/G717R, OVCA1^+/+, and OVCA1^−/− mice were transfected with the reporter plasmid pDual-HIV(−1). Ratios of firefly and renilla luciferase activities were determined 48 h after transfection. (C) CHO cells with various Dph gene deletions were analyzed as in B. CHO WTP4 is the parental line of Dph1-deficient mutant CHO WTP4(Δdph1). CHO K1 is the parental line of the other CHO mutant cells. The results shown are representative of three individual experiments with similar results. (D) Graphical summary of the physiological functions of the diphthamide modification on eEF2.