Slow and steady wins the race: uniting eEF2K, translation and lifespan

Out of all of Aesop’s Fables that I know, which I have now realised isn’t that many, “The tortoise and the hare” was always my favourite as a kid. Whilst the fable has been interpreted in different ways, I always took it to mean that slow and steady wins the race. But what race are we talking about? What about a lifespan race? Yeah, okay, a bit of a tangent, but turns out taking translation a bit more slowly, improving fidelity of protein production, is linked to lifespan in C.elegans. This was elegantly mapped out in the recent Current Biology paper by Xie et. al 1.

slow wins

Figure 1: The tortoise (a.k.a ribosome on the right) taking it slow wins the race. The hare (a.k.a ribosome on the left) at its faster pace makes a mistake (red) in amino acid incorporation/hare sleeping on the job!? …I think I’ve taken this analogy too far.

The elongation phase of translation

Translation of mRNA into protein can be split into three phases; initiation, elongation and termination. Now, so much attention is given to the initiation of translation, which undoubtedly is still important, that it can be easy to forget that the rest of translation must still occur (at least I do anyway). In particular, translation elongation, which is where the bulk of the protein is actually synthesised! But handy for us, the main proteins involved in translation elongation have very “does what is says on the tin” names… the eukaryotic elongation factors (eEFs). eEF1A is responsible for bringing in aminoacyl-tRNAs to the A-site of ribosomes and eEF2 then facilitates translocation of the tRNA into the P-site. eEF2 is regulated via phosphorylation and is phosphorylated by the also well-named eEF2-kinase (eEF2K). The addition of phosphate to eEF2 reduces the affinity of the protein to the ribosome. This reduced eEF2 recruitment slows down the rate of translocation and therefore also the rate of translation.

Since eEF2 is used for protein synthesis of many, if not all nuclear encoded proteins, regulating the activity is very important since it would globally affect protein production. The same goes for eEF2K. So, it may not be a surprise that eEF2K activity is also regulated. This time by a key regulator of general translation in a cell (and ageing), mTORC1 (mammalian target of rapamycin).

mTORC1 activity inhibits eEF2K activity, thus eEF2 is un-phosphorylated and translation is as usual. Lack of mTORC1 activity/activation of eEF2K results in phosphorylation of eEF2 and slowed translation. But why have I told you all this?

The proof is in the lack of pudding

The current opinion is that translation rate can alter the accuracy of translation – faster rates being more likely to mismatch a tRNA-anticodon to a codon and alter the amino acid in the protein. Since eEF2 phosphorylation can slow the rate of translation, is proofing improved in these conditions? It is interesting to point out here that eEF2 doesn’t directly alter the speed of the tRNA-anticodon-codon recognition step, but the translocation step, so would you expect to see a change in misrecognition depending on the phosphorylation state of eEF2? Well, puzzle no longer, Xie’s team tested it.

Using human cells and Firefly luciferase (Fluc) as a translational read-out (and normalising with Renilla luciferase by the dual luciferase technique), two different methods were used to alter eEF2K activity.

Method 1: Deplete eEF2K activity by expressing an siRNA to the gene that is inducible via IPTG.

Method 2: Grow the cells in 2-deoxyglucose (2-DG). This energy-depleted (or “lack of pudding”) environment prevents activation of mTORC1 and hence activates eEF2K.

Cool, but how does this help examine translation accuracy?

Fluc was mutated either by switching arginine for serine (R218S) or adding a “pre-mature” stop codon, both rendering Fluc inactive. However, if the serine codon was mistakenly read as the arginine codon, or the stop codon was read-through then active Fluc would be synthesised. Therefore, detection of Fluc activity in these mutated constructs compared to standard Fluc provides a measurement of translational accuracy. So what did they see?

When eEF2K activity was reduced (e.g by adding IPTG) more errors were made and Fluc activity increased. In the same manner, when IPTG and 2-DG were combined, Fluc levels were greater than with only 2-DG addition (condition where eEF2K is more active). Swapping 2-DG for mTORC1 inhibitors such as rapamycin had similar impact on Fluc expression.

“The worm and the….eEF2K-mutated worm”!?

Since it was known that translational accuracy is correlated with lifespan in many organisms 2, could the activity of eEF2K be playing a role?

Due to their short lifespan and the fact that they possess an orthologue of eEF2K, C.elegans (the worms) was a perfect model to test this. Lifespan can be increased in C.elegans by caloric restriction which reduces activity of TOR (the non-mammalian mammalian TOR), and based on what has been covered so far would be in conditions with greater eEF2K activity.

So what happens to lifespan when caloric restriction and knock out efk-1 (the worm eEF2K) is combined? The answer – no lifespan extension was seen. This suggests that the action of eEF2K is needed for lifespan extension mediated by caloric restriction.

So, what’s the moral of the story?

…Ask good biological questions and design your experiments well with appropriate controls and get awesome findings such as we have seen here. On a more serious note, this work provides further support for the role of proteostasis in lifespan and therefore also likely, healthspan and so promoting eEF2K activity could be a new target to achieve this.

Further reading

  1. Xie, J. et al. Regulation of the Elongation Phase of Protein Synthesis Enhances Translation Accuracy and Modulates Lifespan. Curr. Biol. (2019). doi:10.1016/j.cub.2019.01.029
  2. Ke, Z. et al. Translation fidelity coevolves with longevity. Aging Cell (2017). doi:10.1111/acel.12628

 

P.s I am blaming my eccentricity in this blog to me suppressing it so much to write my dissertation..

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