Maintaining accuracy during protein synthesis is crucial to avoid producing misfolded and/or non-functional proteins. The target of rapamycin complex 1 (TORC1) pathway and the activity of the protein synthesis machinery are known to negatively regulate lifespan in many organisms, although the precise mechanisms involved remain unclear. Mammalian TORC1 signaling accelerates the elongation stage of protein synthesis by inactivating eukaryotic elongation factor 2 kinase (eEF2K), which, when active, phosphorylates and inhibits eEF2, which mediates the movement of ribosomes along mRNAs, thereby slowing down the rate of elongation. We show that eEF2K enhances the accuracy of protein synthesis under a range of conditions and in several cell types. For example, our data reveal it links mammalian (m)TORC1 signaling to the accuracy of translation. Activation of eEF2K decreases misreading or termination readthrough errors during elongation, whereas knocking down or knocking out eEF2K increases their frequency. eEF2K also promotes the correct recognition of start codons in mRNAs. Reduced translational fidelity is known to correlate with shorter lifespan. Consistent with this, deletion of the eEF2K ortholog or other factors implicated in translation fidelity in Caenorhabditis elegans decreases lifespan, and eEF2K is required for lifespan extension induced by nutrient restriction. Our data uncover a novel mechanism linking nutrient supply, mTORC1 signaling, and the elongation stage of protein synthesis, which enhances the accuracy of protein synthesis. Our data also indicate that modulating translation elongation and its fidelity affects lifespan.

Inhibition of (m)TORC1 as well as controlled nutritional restriction are known to extend lifespan in a variety of organisms []. We have therefore also explored the impact of this regulatory system on lifespan; deletion of the eEF2K ortholog reduces the lifespan of C. elegans. Taken together, our data reveal a crucial role for eEF2K in reducing errors in translation, and that reducing translation elongation rates improves translation fidelity and extends lifespan.

Given that mTORC1 controls the activity of eEF2K and thus elongation, we asked whether the effects of mTORC1 signaling on fidelity involve the regulation of eEF2 by eEF2K. Surprisingly, given that eEF2 is not involved in the step of elongation where cognate aminoacyl-tRNAs are selected, we find activation of eEF2K enhances translational fidelity, indicating that its activity nonetheless governs the accuracy of elongation.

The phosphorylation of eEF2 on Thr56 decreases its affinity for the ribosome, thereby inactivating it []. eEF2 is phosphorylated by a dedicated protein kinase, eEF2 kinase (eEF2K), which belongs to the atypical α-kinase family and is subject to tight regulation []. For example, it is inactivated by anabolic and mitogenic signaling pathways such as mTORC1 and the classical extracellular signal-regulated kinase (ERK) MAP kinase pathway []. Thus, activation of these anabolic or mitogenic signaling pathways turns off eEF2K, ensuring that eEF2 and elongation are active. Consistent with this, when initiating ribosomes are stalled using harringtonine, TSC2 null cells (in which mTORC1 signaling is constitutively activated []) exhibit earlier ribosomal runoff, indicating a faster rate of translation elongation (reflecting disinhibition of eEF2), whereas rapamycin—which inhibits mTORC1—slows down elongation []. Conversely, eEF2K is activated by Caions at low pH and under various stress conditions [].

The ribosome then undergoes translocation, whereby it moves the equivalent of one codon along the mRNA, thereby bringing the peptidyl-tRNA into the P site and the next codon into the now-vacant A site. This step requires a second elongation factor, eEF2, whose activity is inhibited by phosphorylation, which is in turn controlled by mTORC1 signaling [].

During mRNA translation, aminoacyl-tRNAs are recruited into the ribosomal A site by eukaryotic elongation factor 1A (eEF1A). After achieving an appropriate codon:anticodon match, the corresponding amino acid is added to the growing polypeptide by forming a peptide bond and concomitant transfer of the nascent chain to the A site tRNA. There is no known connection between mTORC1 signaling and regulation of this step in elongation.

With respect to protein production, maintaining accuracy during protein synthesis (mRNA translation) is crucial for the faithful decoding of genetic information to generate fully functional proteins. However, mRNA translation has a higher intrinsic error rate than DNA polymerase []. Misincorporated amino acids are subject to “editing” in bacteria [], but eukaryotes do not appear to have an analogous function for editing after decoding []. It has been shown that in mammals, as an evolutionary phenomenon, high levels of translational accuracy correlate positively with longer lifespan []. It therefore follows that cells require mechanisms to optimize the accuracy of protein synthesis without compromising the efficiency of protein production. Interestingly, one of the major pathways that modulates accelerated aging in eukaryotic organisms, target of rapamycin complex 1 (TORC1) signaling [], also activates protein synthesis and other anabolic processes []. Recently, Conn and Qian [] showed that active mammalian (m)TORC1 signaling impairs translational accuracy (also termed “fidelity”). Errors in amino acylation of tRNAs and codon decoding can each affect accuracy. Given that >99% of amino acids are incorporated during elongation, the vast majority of translation accuracy errors are made during this stage of translation [].

Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality.

The production of properly made and folded proteins is crucial for normal cell function and organismal survival. Conversely, misfolded proteins can lead to a number of common and serious diseases, including several neurodegenerative disorders [].

It has previously been shown that inhibition of TOR in C. elegans extends lifespan []. Here, we observed that rapamycin-treated efk-1 KO worms still outlived rapamycin-treated WT worms ( Figure 6 D), implying that efk-1 positively contributes to longevity in a TOR-independent fashion in nematodes. As a readout of endoplasmic reticulum (ER) stress, we monitored the levels of P-eIF2α in WT and efk-1 KO worms under normal feeding in the absence or presence of rapamycin or under caloric restriction. Caloric restriction greatly reduced the levels of P-eIF2α, whereas rapamycin also slightly decreased the levels of P-eIF2α in both WT and efk-1 KO worms ( Figure S7 C). However, levels of P-eIF2α were similar in efk-1 KO worms ( Figure S7 C), which corroborates our observations that eEF2K knockdown in A549 cells did not induce phosphorylation of eIF2α ( Figure S5 E). Therefore, the observed impairment of lifespan as the result of efk-1 KO is unlikely to be caused by an increase in ER stress.

C. elegans has an ortholog of eEF2K; its target residue in eEF2 and the adjacent sequence are conserved in C. elegans eEF2 []. As expected, caloric restriction (achieved by using heat-killed [HK] E. coli as food) extended the lifespan of WT N2 worms ( Figure 6 B). efk-1 KO worms (0k3609) (previously characterized in []) had only a slightly reduced lifespan on normal diet but, importantly, showed no extension upon caloric restriction ( Figure 6 B). These data indicate that eEF2K is required for lifespan extension induced by nutrient restriction. Interestingly, depletion of mrs-1 but not of other aminoacyl-tRNA synthetases (lrs-1, kars-1, and nrs-1) further reduced the lifespan of efk-1 KO worms under normal (OP50) feeding ( Figure 6 C). This likely reflects the fact that the latter three enzymes are only required for elongation, which is controlled by eEF2K, whereas mrs-1 is also needed for translation initiation.

mRNA translation accuracy strongly correlates with lifespan of organisms during evolution []. Mammals with high rates of translation fidelity live longer than those with high levels of translation inaccuracy []. We argued that inadequate levels of aminoacyl-tRNA synthetases are expected to impair fidelity by limiting the availability of specific aminoacyl-tRNAs, leading to misincorporation of amino acids at the codons normally recognized by those charged tRNAs (due to use of a near-cognate but incorrect aminoacyl-tRNA). Similar findings were recently reported in yeast []. To test this, we knocked down selected aminoacyl-tRNA synthetases in C. elegans, a short-lived nematode worm that is widely used to study the control of lifespan. (Because, unlike eEF2K, these are essential genes, they cannot be completely knocked out.) Depletion of the leucyl- (lrs-1), arginyl- (rrs-1), asparaginyl- (nrs-1), or methionyl-tRNA (mrs-1) synthetases significantly decreased lifespan when worms were maintained at 25°C on normal food, implying that impaired translation fidelity does indeed shorten lifespan ( Figure 6 A). Drosophila is a widely used and tractable model for studying lifespan, but lacks an ortholog of eEF2K. We found that when leucyl-tRNA synthetase (LeuRS) was knocked down during adulthood in Drosophila, there was a marked reduction in lifespan ( Figures S7 A and S7B), analogous to our findings in C. elegans ( Figure 6 A).

(D) The lifespan of N2 and efk-1 worms when fed with normal food (OP50) containing 100 μM rapamycin (Rap) where indicated. Mean survival: N2/OP50, 12 days; N2/OP50/Rap, 18 days; efk-1/OP50, 10 days; efk-1/OP50/Rap, 14 days. efk-1/OP50 versus efk-1/OP50 + Rap: p < 0.0001; N2/OP50 versus efk-1/OP50: p = 0.0006.

(B) The lifespan of N2 and efk-1 knockout worms when fed with normal food or under caloric restriction (fed with heat-killed [HK] E. coli). Mean survival: N2/OP50 (fed normally with E. coli OP50), 11 days; N2/HK, 12 days; efk-1/OP50, 9 days; efk-1/HK, 9 days. N2/OP50 versus N2/HK: p = 0.0146; N2/HK versus efk-1/ HK: p = 0.0358; N2/OP50 versus efk-1/OP50: p = 0.0006.

Taken together, these data imply that the increases in active Fluc seen in the absence of eEF2K are not due to changes in protein folding or proteasome activity.

To study the effect of altered eEF2K levels on the ubiquitin-proteasome system, we treated A549 and HCT116 cells with the proteasome inhibitor MG132. Increasing concentrations of MG132 evoked phosphorylation of eEF2, indicating activation of eEF2K, in parallel with an increase in polyubiquitinated proteins ( Figures S6 A–S6C). Knocking down eEF2K did not lead to obvious changes in polyubiquitin signals or WT StaCFluc expression in A549 or HCT116 cells ( Figures S6 B and S6C). We also applied a cell-based proteasome assay to measure its chymotrypsin-like activity in these cells ( Figures S6 D–S6G). We observed decreased proteasome activity in 2-DG-treated A549 cells ( Figure S6 D) and increased proteasome activity in A549 cells that had been incubated in DPBS ( Figure S6 G). However, under no conditions tested did knockdown of eEF2K affect proteasome activity ( Figures S6 D–S6G).

It was important to assess whether changes in chaperone or proteasome activity contributed to the increase in active Fluc that we observe when knocking down eEF2K. In vitro Fluc-refolding assays revealed that there was a slight decrease in refolding ability when denatured recombinant Fluc was incubated with lysates from eEF2K knockdown A549 cells that had been pre-treated with 2-DG, pH 6.7 medium, or AZD8055 ( Figures S5 K–S5N) as compared to lysates from control cells with normal eEF2K levels. The reduced refolding capacity might reflect higher levels of client misfolded proteins resulting from impaired translation fidelity in the knockdown cells. The rise in active Fluc levels upon eEF2K knockdown cannot therefore be explained by lower protein folding or chaperone capacity; indeed, these data may indicate that the measured effects of eEF2K to promote translation fidelity are a slight underestimate.

Treating the cells with 2-DG to increase eEF2 phosphorylation tended to reduce the expression of Fluc. Knocking down eEF2K significantly increased the expression of active Fluc from GUG or CUG as start codons but did not affect expression of Fluc with AUG as start codon ( Figure 5 B). This implies that increased phosphorylation of eEF2, and thus slower elongation, also aids the fidelity of start codon recognition, by augmenting the ability of ribosomes to distinguish between AUG and near-cognate start codons ( Figure 5 C).

We first compared the relative expression levels of luciferase expressed from these plasmids in A549 cells. Non-AUG start codon plasmids exhibited 7.7% (GUG) or 17.3% (CUG) of Fluc activity compared to the WT Fluc (AUG start codon with optimal Kozak consensus; Figure 5 A).

(C) Schematic representation of how slow elongation speed favors the correct selection of the start codon as well as of the cognate codon:anticodon (tRNA) pair.

(B) A549 cells were transfected with pICtest2 reporter vectors as in (A), and after 48 h were treated with 2-DG as in Figures 1 D–1F. Fluc activity was measured and is expressed as percentage of control (means ± SD; n = 4 independent experiments, each performed in triplicate).

(A) A549 cells were transfected with pICtest2 vectors encoding StaCFluc with different start codons (previously reported in []; sequences are indicated). Fluc activity was then measured, and is expressed as percentage of control (GCCAUGG) (means ± SD; n = 4 independent experiments conducted in triplicate). 0.01 ≤p < 0.05,p < 0.001, as obtained by one-way ANOVA followed by Dunnett’s test.

We previously [] developed “pICtest2” dual-luciferase reporters with non-AUG start codons as well as an inferior Kozak consensus (non-optimal initiation [start]-site context). To test the effect of eEF2K on start-site recognition, we selected two near-cognate non-AUG start codon variants (GUG and CUG).

The process of start-site selection in eukaryotes differs fundamentally from aminoacyl-tRNA choice during elongation; in particular, during initiation, the 40S subunit (with its associated factors) is already equipped with the initiator methionyl-tRNA and is seeking the correct codon, whereas in elongation the converse is true: a correct match has to be achieved between the codon already in the A site and the relevant aminoacyl-tRNA. Chu et al. [] reported that in yeast, when rare (infrequently used) codons are positioned immediately after the translation start codon, the impaired speed of translation elongation restricts the “liberation” of the 40S ribosome from the start codon and hence loading of the next 40S subunit. Under these circumstances, translation elongation can affect initiation. However, it was not known whether this also applies in mammalian cells. Because eEF2K regulates the rate of elongation, we asked whether eEF2K affected the stringency of translation start-site selection.

Our data indicate that stimulation of eEF2K, and thus lower eEF2 activity, promotes translational accuracy. It follows that reducing eEF2 protein levels should also improve fidelity. Accordingly, A549 cells were transfected with siRNA against eEF2 and, 24 h later, with the vectors encoding Fluc or mutants. After a further 48 h, by which time eEF2 levels had been reduced substantially ( Figure 4 D), luciferase activity was measured. Knockdown of eEF2 decreased the levels of active luciferase from the StaCFluc[R218S] and StaCFluc[STOP] vectors but had no effect on WT StaCFluc expression ( Figure 4 E). Taken together, these data indicate that the AMPK-eEF2K-eEF2 pathway is crucial for translation quality control when cells are subjected to stress conditions.

For experiments where cells were treated with 2-DG, they were transferred into medium containing lower glucose levels (5.5 mM). Even without 2-DG, this resulted in increased p-eEF2 levels ( Figures S3 and S4 ). This likely explains why we observed an increase in Fluc activity in Fluc[R218S]/Fluc[STOP]-transfected or eEF2K knockdown A549 or HCT116 cells ( Figures 1 E, S2 E, and S3 ), as well as in AMPKMEFs ( Figure 4 C), when compared to the corresponding control cells.

Importantly, compared to WT MEFs, AMPK null MEFs transfected with the Fluc vector exhibited higher levels of Fluc activity from the StaCFluc[R218S] and StaCluc[STOP] but not from the StaCFluc[WT] vectors ( Figure 4 C), illustrating that AMPK helps ensure translational accuracy under stress conditions. Given that AMPK activates eEF2K, and the above data linking eEF2K to translational accuracy, these effects are likely to be mediated via activation of eEF2K and concomitant inhibition of eEF2.

Stress conditions can evoke activation of eEF2K via the AMPK. Genetic KO of AMPK completely (2-DG; Figure 4 A) or partially (AZD8055, DPBS, low pH; Figure 4 B) prevented increased phosphorylation of eEF2, indicating that AMPK plays a role in mediating these effects. In line with our previous studies [], we also observed a reduction in levels of eEF2K protein in AMPK null MEFs ( Figures 4 A and 4B); this may well also contribute to the lower eEF2 phosphorylation seen in these cells.

Results are given as means ± SD; n = 4 independent experiments performed in duplicate. ∗ 0.01 ≤ p < 0.05, ∗∗ 0.001 ≤ p < 0.01, ∗∗∗ p < 0.001, as obtained by two-way ANOVA.

(D) A549 cells were transfected with scrambled siRNA or siRNA against EEF2 for 24 h; cells were then transfected with WT/R218S/STOP StaCFluc. After 48 h, cells were lysed followed by SDS-PAGE/western blotting analysis.

(C) AMPK +/+ and AMPK −/− MEFs were transfected with StaCFluc WT, R218S, or STOP constructs. After 24 h, cells were then placed in low-glucose (5.5 mM) DMEM for 1 h, before being treated with 2-DG (10 mM) for 16 h. Fluc activity was measured and normalized to vehicle-treated control.

(B) AMPK +/+ and AMPK −/− MEFs were cultured for 3 h with 1 μM AZD8055 or vehicle (DMSO), or in medium buffered at pH 7.4 or 6.7 for 16 h, or growth medium (as control) or DPBS. Cells were then lysed and lysates were subjected to immunoblotting analysis with the indicated antibodies.

(A) AMPK +/+ and AMPK −/− MEFs were pre-incubated in low-glucose (5.5 mM) DMEM for 1 h, before treatment with 2-DG (10 mM) for 16 h.

The treatment of cells with 2-DG, AZD8055, DPBS, or low pH decreased global protein synthesis. Although knocking down eEF2K did not affect overall protein synthesis rates under any of the conditions tested ( Figures S5 G–S5J), it did increase the proportion of translationally inactive monosomal fractions concomitant with a decrease in translationally active polysomal fractions, as analyzed on sucrose density gradients ( Figure 3 ). This presumably reflects faster rates of elongation in the knockdown cells (as fewer ribosomes are engaged in polysomes at any given time, due to ribosomal “run-off”). Hence, the positive effect of eEF2K on translation fidelity reflects slower translation elongation rather than lower overall rates of protein synthesis.

For all experiments, IPTG (1 μM) was added to A549 cells 5 days before experiments to induce the expression of shRNA against EEF2K.

(E) A549 cells were cultured either in growth media or DPBS for 3 h before polysome profile analysis.

(C) A549 cells were cultured under low-glucose (5.5 mM) DMEM for 1 h before the addition of 2-DG (10 μM) for 16 h. Cells were then subjected to polysome profile analysis.

Inducing the shRNA reduced eEF2K levels and attenuated eEF2 phosphorylation ( Figures 2 E, 2F, and S1 I–S1S). Incubation in DPBS modestly decreased StaCFluc-PEST[WT] ( Figure 2 D), whereas hypoxia (5% oxygen) greatly reduced StaCFluc[WT] levels ( Figures 2 E and 2F). Cells cultured in medium buffered at low pH (6.7), in DPBS, or under hypoxia exhibited reduced levels of active Fluc from the StaCFluc, StaCFluc-PEST R218S, or STOP vectors ( Figures 2 C–2F), indicating enhanced translational accuracy. Although knockdown of eEF2K did not affect the expression of StaCFluc/StaCFluc-PEST[WT], it did increase levels of active (mistranslated) StaCFluc/StaCFluc-PEST R218S under these conditions ( Figures 2 C–2F and S5 F). These data provide further evidence that eEF2K serves as a “guardian” of translation fidelity under diverse stress conditions.

We also wished to test whether eEF2K is involved in maintaining the quality of translation under other conditions. We therefore incubated A549 cells, which had been transfected with vectors for StaCFluc[WT] or mutants, under stress conditions known to activate eEF2K, i.e., incubation in medium buffered at low pH (pH 6.7; Figures 2 C, S1 O, and S1P), in DPBS (which lacks amino acids; Figures 2 D, S1 Q, and S1R), or under hypoxia ( Figures 2 E, 2F, and S1 S). All these treatments inhibited signaling through mTORC1, as shown by the decreased rpS6 phosphorylation and increased electrophoretic mobility of 4E-BP1 (reflecting its dephosphorylation) ( Figures S1 I–S1S). Phosphorylation of eIF2α, a key regulator of translation initiation, was not affected, although it was induced by cycloheximide (used as a positive control for the P-eIF2α antibody; Figure S5 E).

Rapamycin or AZD8055 treatment reduced expression of active StaCFluc from StaCFluc/StaCFluc-PEST [R218S] and [STOP] vectors but not from the WT vector, indicating lower rates of misreading or termination readthrough errors. Consistently, knocking down eEF2K using inducible shRNA in A549 cells ( Figures 2 A and S5 D) or KO of eEF2K in MEFs ( Figure 2 B) significantly attenuated the ability of mTOR inhibitors to decrease error rates. Thus, the ability of mTORC1 signaling to modulate error rates requires eEF2K, an effector of mTORC1 signaling. The data show that mTORC1 regulates translation fidelity by affecting the rate of translation elongation via eEF2K, thus providing a clear molecular mechanism to explain the findings of Conn and Qian regarding the impact of mTORC1 signaling on translation fidelity [].

Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality.

Active mTORC1 signaling increases global protein synthesis but impairs translational fidelity []. Because mTORC1 is an upstream negative regulator of eEF2K [], we asked whether eEF2K mediates the effect of mTORC1 on translational fidelity. For this purpose, we incubated A549 cells ( Figures 2 A, S1 K, and S1M) as well as eEF2Kand eEF2KMEFs ( Figures 2 B, S1 L, and S1N) in the presence or absence of rapamycin or AZD8055 []. As expected, rapamycin or AZD8055 treatment abolished the phosphorylation of rpS6 at Ser240/Ser244, indicating mTORC1 was inhibited, and increased the phosphorylation of eEF2 at Thr56 ( Figures S1 K–S1N). Genetic deletion of eEF2K totally blocked phosphorylation of eEF2 ( Figure S1 N).

For experiments performed in A549 (A and C–E) and HCT116 (F) cells, IPTG (1 μM) was added to cells 5 days before StaCFluc transfection to induce the expression of shRNA against EEF2K. Fluc activity is expressed as means ± SD; n = 4 (A–D) or 3 (E and F) independent experiments performed in triplicate. See also Figures S1 S2 , and S4–S6

(E and F) A549 (E) or HCT116 (F) cells were transfected with StaCFluc WT, R218S, or STOP constructs. After 24 h, they were incubated in 20% or 5% O 2 for 16 h, before luciferase assay.

(D) A549 cells were transfected with Fluc-PEST WT, R218S, or STOP constructs, followed by incubation in growth medium or DPBS for 3 h, before luciferase assay analysis.

(C) A549 cells were transfected with StaCFluc WT, R218S, or STOP constructs. 24 h later, cells were incubated in pH (7.4 or 6.7) buffered medium for 16 h, before luciferase assay analysis.

(B) eEF2K +/+ and eEF2K −/− MEFs were transfected with StaCFluc WT, R218S, or STOP, and then incubated with vehicle (DMSO), rapamycin (200 nM), or AZD8055 (1 μM). After 16 h, Fluc activity was determined using luciferase assay.

(A) A549 cells were transfected with StaCFluc WT, R218S, or STOP constructs. After 24 h, cells were treated with vehicle (DMSO, as a control), rapamycin (200 nM), or AZD8055 (1 μM) for 16 h. After 16 h, Fluc activity was determined using luciferase assay.

AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.

Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality.

Aminoacyl-tRNA synthetases (aaRSs) play a crucial role in protein synthesis and in maintaining its fidelity by generating correctly charged tRNAs. Errors during elongation may arise from competition of cognate and near-cognate aminoacyl-tRNAs at the ribosomal A site; therefore, lower levels of the cognate aminoacyl-tRNA are expected to give near-cognate aminoacyl-tRNAs a higher chance of being accepted at a given codon []. Consistent with this, and as expected from the nature of the mutation in this vector, expression of StaCFluc[R218S] was greatly enhanced in cells where expression of the seryl aminoacyl-tRNA synthetases (SARS) was knocked down by small interfering RNA (siRNA) ( Figures 1 G–1I). The observed magnitude of the change may be an underestimate, because SARS knockdown led to a reduction in the expression of StaCFluc[WT].

We also used CRISPR/Cas9 genome editing to generate eEF2K knockout (KO) MDA-MB-231 (human breast cancer) cells. Whereas incubation in 2-DG slowly increased p-eEF2 levels in WT cells ( Figure S5 A), no eEF2 phosphorylation was seen in KO cells ( Figure S5 B). We observed an increase in the expression of active StaCFluc from StaCFluc[R218S]-transfected eEF2K-KO cells as compared to control cells ( Figure S5 C), similar to our observations for A549 and HCT116 cells where EEF2K was knocked down by inducible shRNA ( Figures 1 D–1F and S3 ). Fluc mRNA abundance ( Figures S4 A–S4D) as well as Fluc protein expression levels ( Figures S4 E–S4K) were very similar in WT and IPTG-treated A549 cells and in eEF2Kand eEF2Kmouse embryonic fibroblasts (MEFs); thus, the differences in Fluc activity observed ( Figures 1 S2 E, and S3 ) were not due to a difference in transfection, transcription, or translation efficiency. Levels of total Fluc from the WT or R218S vectors were very similar, indicating that the differences in activity reflect mistranslation in the case of the latter ( Figure S4 E). (Note that MinCFluc [ Figure S1 I] and Fluc[STOP] [ Figure S4 E] were not detected by the Fluc antibody, in the former case as levels were too low and in the latter probably because either the epitope lies after the stop codon or the truncated Fluc is rapidly degraded.)

To test the role of eEF2K in translational accuracy, we made use of A549 or HCT116 cells that inducibly express a short hairpin (sh)RNA that targets the EEF2K mRNA ( Figures 1 D–1F and S1 K–S1N) []. We transfected them with vectors for wild-type (WT) or mutant StaCFluc. In some cases, cells were treated with isopropyl β-D-1-thiogalactopyranoside (IPTG) to induce the shRNA against EEF2K (1–4). Cells were then cultured for 16 h in the presence or absence of 2-DG. Knocking down eEF2K by inducible shRNA did not affect the Fluc/Rluc activity ratio from the Min/Sta/MaxCFluc[WT] or StaCFluc[WT-PEST] vectors ( Figures 1 E, 1F, S2 E, S3 , and S4 ). However, knockdown of eEF2K did increase expression of active StaCFluc from the StaCFluc[R218S-PEST] and StaCFluc[STOP-PEST] vectors ( Figures S3 C–S3E), as well as Min/Sta/MaxCFluc[R218S] and Min/Sta/MaxCFluc[STOP] ( Figures 1 D–1F and S3 ) either under basal conditions (cells cultured in DMEM containing low glucose [5.5 mM]) or when cells were treated with 2-DG.

Because eEF2K regulates the rate of elongation, we wished to explore its role in modulating translational fidelity. As noted above, eEF2K is activated under diverse stress conditions []. Using A549 (human lung adenocarcinoma) cells, we first studied the effects of (1) energy deprivation, mimicked by treating cells with 2-deoxyglucose (2-DG), a glucose analog that can be phosphorylated by hexokinase but cannot be metabolized through glycolysis; consequently, it depletes cells of ATP; (2) nutrient deprivation (cells were transferred to Dulbecco’s [D-]PBS); and (3) extracellular acidosis, which activates eEF2K []. We also treated cells (4) with the mTOR inhibitor AZD8055 [], which alleviates inhibitory inputs into eEF2K ( Figures S1 K–S1S and S2 A–S2D). As expected, each of these conditions inhibited mTORC1 signaling, as shown by decreased rpS6 phosphorylation (downstream of mTORC1), and evoked activation of eEF2K, as indicated by increased eEF2 phosphorylation ( Figures S1 K–S1S and S2 A–S2D). Notably, in A549 cells, 2-DG treatment rapidly induced the phosphorylation of eEF2 (within 30 min), which persisted for at least 16 h ( Figures S2 A–S2D).

AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity.

Importantly, we observed the highest levels of these translation errors (higher luciferase activity) in HEK293 cells transfected with the hMaxCFluc mutants, whereas the lowest levels of FLuc, and therefore of errors, were seen in cells transfected with the hMinCFluc mutants ( Figure 1 C; note that data are always compared to the differing yields of active luciferase for the corresponding parent Min, Sta, or Max vectors). These results show that faster translation elongation leads to impaired translational fidelity.

In yeast, bacterial [], and mammalian [] cells, faster elongation rates compromise the accuracy of codon:anticodon recognition, although it has recently been reported that rates of translation elongation do not correlate with translation fidelity in yeast []. The above constructs allow us to assess the extent to which the rate of elongation affects the accuracy (fidelity) of mRNA translation, and explore what factors affect this. To do this, we created two Fluc reporter missense mutants for use in mammalian cells. In the first, the arginine codon (AGA) at position 218 was changed to a serine codon (AGT), yielding the “Fluc[R218S]” vectors. Because Arg218 is a key residue for binding of substrate to luciferase [], correct decoding at this position generates catalytically inactive protein. The level of active luciferase (in which this codon has actually been misread as encoding arginine) therefore serves as an indicator of misreading of the serine codon to incorporate arginine, i.e., lack of fidelity during elongation []. In the second variant, the leucine codon (CTT) at position 210 of the corresponding amino acid sequence was replaced by a stop codon (TGA, yielding the Fluc[STOP] vector), allowing us to detect readthrough errors during translation [], i.e., another failure in its accuracy. Because changes in steady-state protein levels could, in principle, also result from altered rates of protein degradation, we made the same mutations in a StaCFluc reporter that encodes Fluc followed by a PEST motif (SSGTRHGFPPEVEEQAAGTLPMSCSQESGMDRHPAACASARINV). This motif causes the destabilization and rapid degradation of newly synthesized Fluc (t≈ 2 h) [] (vectors termed Fluc[R218S-PEST] and Fluc[STOP-PEST], respectively; see Figure S1 E). This accelerates the turnover of Fluc, allowing us to more accurately assess new Fluc protein production without interference from already-existing enzyme present at the start of the treatment.

Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality.

The rate of translation elongation can be affected by the codon composition of the translated region of mRNAs; because some codons are used less often than others and hence there are fewer tRNAs with the corresponding anticodon, it takes longer to achieve a codon:anticodon match for such codons. This can therefore slow down the decoding of the mRNA []. Previously, constructs were created encoding firefly luciferase (Fluc; see Table 1 for a list of specific terms used in this study) using the slowest (yeast MinCFluc) and fastest (yeast MaxCFluc) possible codons []. Similarly, and to assess whether codon usage modulates synthesis rates in human cells, we made the slowest (human [h]MinCFluc) and fastest (hMaxCFluc) constructs according to predicted human decoding times ( Figures 1 A and S1 A–S1J) in a vector that also contains a cistron encoding Renilla luciferase (Rluc); Rluc activity levels allow the Fluc data to be normalized to control for factors such as transfection efficiency or overall translational activity. All data are therefore given as “relative Fluc activity,” i.e., Fluc/Rluc ratio as a % of the corresponding control conditions. We named the original wild-type “standard” Fluc “StaCFluc.” hMaxCFluc produced the highest amount of active Fluc protein, whereas hMinCFluc produced much less ( Figures 1 B, S1 I, and S1J), showing that codon composition strongly affects the overall efficiency of protein expression and thereby demonstrating that elongation rates limit the overall rate of translation of this mRNA.

Results are given as means ± SD.0.01 ≤ p < 0.05,0.001 ≤ p < 0.01,p < 0.001, as determined by two-way ANOVA followed by Dunnett’s test (A) or Tukey’s test (C); n = 4 (B and C). See also Figures S1 and S3–S5

(I) A549 cells were transfected with scrambled or SARS siRNAs. After 24 h, the cells were transfected with StaCFluc WT or R218S vectors. 48 h later, luciferase activity was quantified. Results are given as means ± SD; n = 4 independent experiments, each performed in triplicate.

(G) A549 cells were transfected with scrambled or SARS siRNA. After 72 h, they were lysed, and samples were subjected to immunoblotting analysis with the indicated antibodies.

(E and F) IPTG (1 μM) was added to A549 cells 5 days before the experiment to induce expression of an shRNA against EEF2K. A549 cells were transfected with StaCFluc (E)/MaxCFluc (F) WT, R218S, or STOP constructs, and cells were then cultured in DMEM containing low glucose (5.5 mM) for 1 h, before treatment with 2-DG (10 mM) for 16 h. Fluc activity was quantified using luciferase assay and normalized to mock-treated controls. Results are given as means ± SD; n = 3 independent experiments, each performed in triplicate.

(D) A549 cells were treated with 1 μM IPTG for up to 120 h (5 days) to induce the expression of an shRNA against EEF2K.

(C) HEK293 cells were transfected with the MinCFluc WT, R218S, or STOP; StaCFluc WT, R218S, or STOP; or MaxCFluc WT, R218S, or STOP constructs. After 48 h, relative Fluc activity was then analyzed using luciferase assay. The relative Fluc expression levels when using R218S or STOP constructs were then normalized and presented as % of WT control.

(B) HEK293 cells were transfected with empty pICtest2 or pICtest2 vectors encoding Min/Sta/MaxCFluc. After 48 h, Fluc activity was quantified using luciferase assay.

(A) The heatmap illustrates the calculated decoding speed of the designed Min/Sta/MaxCFluc constructs (a color bar is shown below for reference).

Discussion

5 Drummond D.A.

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Wilke C.O. Mistranslation-induced protein misfolding as a dominant constraint on coding-sequence evolution. 35 Orgel L.E. Ageing of clones of mammalian cells. 36 Orgel L.E. The maintenance of the accuracy of protein synthesis and its relevance to ageing: a correction. 37 Orgel L.E. The maintenance of the accuracy of protein synthesis and its relevance to ageing. 38 Edelmann P.

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Goldstein S. Fidelity of protein synthesis does not decline during aging of cultured human fibroblasts. 40 Mori N.

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Goto S. Codon recognition fidelity of ribosomes at the first and second positions does not decrease during aging. 41 Szajnert M.F.

Schapira F. Properties of purified tyrosine aminotransferase from adult and senescent rat liver. 5 Drummond D.A.

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Gorbunova V. Translation fidelity coevolves with longevity. 6 Ke Z.

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Gorbunova V. Translation fidelity coevolves with longevity. The faithful translation of mRNAs into proteins plays crucial roles in normal cell functions and organismal lifespan. During translation, amino acid misincorporation is estimated to happen at a rate between every 1 in 1,000 and 10,000 codons []. Over time, even low error rates in the synthesis of certain proteins, such as disease-associated ones, e.g., SOD1 (whose misfolding causes amyotrophic lateral sclerosis), can lead to accumulation of defective proteins even affecting the faithful transmission of genetic information []. In the 1960s–1970s, Orgel [] proposed that increased rates of translation errors directly affect aging and lifespan. Although this theory was challenged in the 1970s–1980s [], it was recently demonstrated that mRNA translation accuracy imposes strong evolutionary pressure []. Indeed, mammals with low levels of translation inaccuracy live longer than those with high rates of translation errors [].

42 Harrison D.E.

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Kapahi P. 4E-BP extends lifespan upon dietary restriction by enhancing mitochondrial activity in Drosophila. 45 Syntichaki P.

Troulinaki K.

Tavernarakis N. eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans. 43 Selman C.

Tullet J.M.

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et al. Ribosomal protein S6 kinase 1 signaling regulates mammalian life span. 7 Zoncu R.

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Sabatini D.M. mTOR: from growth signal integration to cancer, diabetes and ageing. 46 Hansen M.

Taubert S.

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Lee S.J.

Kenyon C. Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Decades of studies have shown that caloric restriction can extend lifespan in invertebrates and in rodents. More recent studies have implicated key cellular components linked to the control of translation and that are regulated (directly or indirectly) by nutrients in modulating lifespan. These include mTORC1 [] and its downstream substrates or effectors S6K1 ([]; in mice), 4E-BP1 ([]; in D. melanogaster), eIF4E ([]; in C. elegans), and AMPK ([]; in mice). Inhibition of mTOR signaling (a pathway that activates protein synthesis) extends lifespan in diverse organisms from yeast to mammals []. Furthermore, a range of conditions that slow down translation all extend lifespan in C. elegans [].

9 Conn C.S.

Qian S.B. Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality. 15 Inoki K.

Li Y.

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Wu J.

Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. 9 Conn C.S.

Qian S.B. Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality. 9 Conn C.S.

Qian S.B. Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality. Translation fidelity is assured by two main events: synthesis of the precisely paired aminoacyl-tRNAs by aminoacyl-tRNA synthetases, and the stringent selection of proper aminoacyl-tRNAs by the ribosomes during elongation. It has been shown that translation rates inversely correlate with cotranslation folding efficiency as well as translational accuracy []. Conn and Qian demonstrated that rates of mRNA mistranslation in TSC2 null cells (where mTORC1 signaling is constitutively activated []) are significantly higher than in WT cells []. They also showed that the impairment of translational fidelity in TSC2 null cells results from activation of the mTORC1-S6K1 pathway and that TSC2 null cells exhibit faster translation elongation []. However, prior to this, it was not known whether translation elongation directly impacted on translation fidelity or, if so, how this was linked to the mTORC1 pathway.

27 Leprivier G.

Remke M.

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Mateo A.R.

Kool M.

Agnihotri S.

El-Naggar A.

Yu B.

Somasekharan S.P.

et al. The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation. 28 Xie J.

Mikolajek H.

Pigott C.R.

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Moore C.E.

Mohammed H.

Werner J.M.

Thomas G.J.

Proud C.G. Molecular mechanism for the control of eukaryotic elongation factor 2 kinase by pH: role in cancer cell survival. 29 Moore C.E.

Mikolajek H.

Regufe da Mota S.

Wang X.

Kenney J.W.

Werner J.M.

Proud C.G. Elongation factor 2 kinase is regulated by proline hydroxylation and protects cells during hypoxia. Here, we demonstrate faster translation due to the presence of surrounding codons with high usage rates (“fast codons”) leads to higher levels of mistranslation ( Figures 1 A–1C), whereas slower translation elongation reflecting low usage-rate codons (“slow codons”) correlates with reduced rates of translation errors. This provides further evidence that increased productivity of the translation machinery occurs at the expense of poorer accuracy. Here we demonstrate that, under various stress conditions, where eEF2K is activated and slows down translation [], eEF2K aids translational accuracy. Conversely, when mTORC1 signaling is active and switches off eEF2K to accelerate elongation, this reduces translation fidelity.

14 Wang X.

Regufe da Mota S.

Liu R.

Moore C.E.

Xie J.

Lanucara F.

Agarwala U.

Pyr Dit Ruys S.

Vertommen D.

Rider M.H.

et al. Eukaryotic elongation factor 2 kinase activity is controlled by multiple inputs from oncogenic signaling. 47 Wang X.

Li W.

Williams M.

Terada N.

Alessi D.R.

Proud C.G. Regulation of elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. 9 Conn C.S.

Qian S.B. Nutrient signaling in protein homeostasis: an increase in quantity at the expense of quality. A reduction in eEF2K levels or its inactivation reduces the accuracy of decoding. mTORC1 can directly or indirectly drive the phosphorylation of several residues in eEF2K that impair its activity []; for example, S6K1 directly phosphorylates eEF2K at Ser366 and thereby inhibits its activity []. Importantly, we show that disabling eEF2K attenuates the ability of mTOR inhibitors to promote translational fidelity ( Figures 2 A–2E), thereby providing a molecular mechanism for the ability of mTORC1 to modulate the accuracy of protein synthesis []. Thus, surprisingly, although eEF2 does not itself mediate the step in elongation where aminoacyl-tRNAs are recruited to the A site, where a correct codon:anticodon match must be achieved, it does nevertheless link mTORC1 signaling and thus multiple stress conditions that activate eEF2K to the accuracy of elongation. These conditions include impaired glycolysis ( Figures 1 E and 1F), low pH ( Figure 2 C), nutrient deprivation ( Figure 2 D), and hypoxia ( Figures 2 E and 2F). Because levels of global protein synthesis were unaffected when eEF2K was knocked down ( Figures S5 G–S5J), translation fidelity appears to be affected mainly by the speed of elongation rather than the overall rate of protein production. Thus, our data point to eEF2K-mediated regulation of elongation helping to ensure accuracy rather than affecting overall rate of protein production, at least in the cells and under the conditions tested here.

15 Inoki K.

Li Y.

Zhu T.

Wu J.

Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. 48 Horman S.

Browne G.

Krause U.

Patel J.

Vertommen D.

Bertrand L.

Lavoinne A.

Hue L.

Proud C.

Rider M. Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis. 28 Xie J.

Mikolajek H.

Pigott C.R.

Hooper K.J.

Mellows T.

Moore C.E.

Mohammed H.

Werner J.M.

Thomas G.J.

Proud C.G. Molecular mechanism for the control of eukaryotic elongation factor 2 kinase by pH: role in cancer cell survival. 29 Moore C.E.

Mikolajek H.

Regufe da Mota S.

Wang X.

Kenney J.W.

Werner J.M.

Proud C.G. Elongation factor 2 kinase is regulated by proline hydroxylation and protects cells during hypoxia. AMPK is activated upon lack of metabolic energy and lies upstream of mTORC1 and eEF2K, suppressing or stimulating them, respectively []. We show that the activation of AMPK also plays a positive regulatory role in translation fidelity; AMPK null MEFs exhibit impaired translation accuracy. This may reflect loss of the ability of AMPK to inhibit mTORC1 and stimulate eEF2K and/or the lower eEF2K protein levels in AMPK null cells [] ( Figures 4 A and 4B).

49 Kearse M.G.

Green K.M.

Krans A.

Rodriguez C.M.

Linsalata A.E.

Goldstrohm A.C.

Todd P.K. CGG repeat-associated non-AUG translation utilizes a cap-dependent scanning mechanism of initiation to produce toxic proteins. We also show that activation of eEF2K helps reduce initiation at codons similar to canonical AUG start sites, i.e., CUG or GUG ( Figure 5 ). Initiation involves “scanning” by 40S ribosomal subunits and associated factors to seek appropriate start codons; this is facilitated by the fact that the relevant methionyl-initiator tRNA is already associated with them. Although the presence of secondary structure downstream of a start codon favors initiation from non-AUG codons and contributes to neurodegenerative diseases such as ALS (amyotrophic lateral sclerosis) and FXTAS (fragile X-associated tremor/ataxia syndrome) [], our data actually suggest that impaired elongation, which would slow down ribosomes within the open reading frame, actually favors the “correct” choice of an AUG start codon, possibly because faster elongation (in cells where eEF2K has been knocked down) allows less time for selection of the optimal initiation codon (AUG) pair at the start site and acceptance of otherwise less favored ones such as GUG and CUG ( Figure 5 C).

A further key finding of this study is that efk-1 is required for the lifespan extension that normally occurs in worms under nutritionally restricted conditions. Furthermore, because ablation of several aminoacyl (leucyl, arginyl, asparaginyl, or methionyl)-tRNA synthetases also decreases lifespan in nematodes, our data clearly demonstrate that factors implicated in regulating translational accuracy influence longevity ( Figure 6 ). However, it should be noted that aminoacyl-tRNA synthetases and eEF2K (efk-1 in nematodes) are proteins that are required for or regulate, respectively, mRNA translation. Although each has a specific function, they may exert pleiotropic effects, given the crucial importance of protein synthesis for cell physiology. Therefore, the decreased lifespan we observe in nematodes where efk-1 or aminoacyl-tRNA synthetases were knocked down may be due to these effects. Also, although the correlation between slower elongation and greater lifespan holds true in nematodes and human cells, because TORC1 signaling is not known to regulate eEF2 in C. elegans, additional mechanisms must exist in them to account for the link between mTORC1 and longevity in nematodes.

Our data show that eEF2K promotes the fidelity of translation elongation and initiation, thereby providing a link between inhibition of mTORC1 signaling (which activates eEF2K in mammals) and the enhanced accuracy of protein synthesis. These data provide a mechanism for the very well-known association between impaired mTORC1 signaling or slower protein synthesis and greater lifespan.