Field of Science

Testing predictions from phylogenetics: mRNA decay mechanisms in Archaea

How do you make an bioinformatician with particular interest in protein functional evolution happy? By testing their functional predictions experimentally. Well, that's that's what makes this particular bioinformatician happy anyway! 

In 2008, I had a paper out with Vasili Hauryliuk on the evolution of the Hbs1/eRF3/Ski7 and eRF1/Dom34 protein families, which was published in BMC Evolutionary Biology. eRF3 and Hbs1 belong to the EF1 family of translational GTPases. They orginated from a duplication of eukaryotic elongation factor eEF1A (also found as aEF1A in archaea) before the last common ancestor of all extant eukaryotes.

Schematic diagram of the phylogenetic relationships among the major families of trGTPases. Subfamily names are coded by their taxonomic distribution as follows; green and underlined: bacteria, red and italic: eukaryotes, blue with names prefixed with e/a: subfamilies present in both eukaryotes and archaea. Figure from my conference abstract published in the journal of Molecular Structure and Dynamics

A bit of background is probably in order. eEF1A is responsible for binding and delivering aminoacyl-tRNA (aa-tRNA) to the A site of the ribosome during the elongation stage of protein synthesis. Elongation factor eRF3 acts in an analogous manner during the termination stage, binding and delivering eRF1 to the A site. eRF1 is the stop codon-recognising factor, and is a structural mimic of aa-tRNA. eRF1/3 and participate in an mRNA surveillance machanism called nonsense mediated decay (NMD), promoting mRNA decay in response to premature stop codons. Hbs1 is the paralogue of eRF3 and binds Dom34, itself a paralogue of eRF1. Together, Hbs1 and Dom34 (also called pelota) function in another mRNA surveillance mechanism no-go decay, onset when a ribosome is stalled on the mRNA. Then there's Ski7, which is only found in Saccharomycetale fungi and functions in non-stop decay, where a stop codon fails to be interpreted, and translation runs on through to the poly-A tail of the mRNA. Here's the schematic family tree of these characters:

Evolution of mRNA quality control mechanisms. A schematic representation of a proposed scenario for the origin and divergence of components of trGTPase-associated mRNA decay mechanisms in archaea and eukaryotes

And the actual phylogenetic trees from Bayesian phylogenetic analyses with MrBayes:


One of the most interesting things about this family is that Archaea don't have Hbs1 or eRF3. We proposed two possible explanations for how translation termination, nonsense-mediated and no-go decay work in this domain of life. 1: eRF1 and Dom34 can carry out their functions without the need of a GTPase to deliver them to the ribosome. 2. Another GTPase is doing it, the most likely candidate being the closest relative in archaea: elongation factor 1A (aEF1A). Recently, two exciting papers were published in PNAS that confirmed that scenario number 2 is the case.

In Kobayashi et al., the crystal structure of the aDom34 and GTP-bound aEF1A complex is presented, and biochemical and genetic analyses of Saito et al. show evidence of binding between aRF1 and aEF1A. Using the Kobayashi et al structure, a docking model of the aRF1·aEF1A·GTP complex has also been constructed.

So, with aEF1A being multifunctional in these mechanisms, this suggests that the original eukaryotic eEF1A was too (before the duplications that gave rise to eRF3 and Hbs1p). The modern eEF1A has a whole range of additional "moonlighting" functions (see Ejiri, 2002). Perhaps the evolution of Hbs1 and eRF3 freed up eEF1A from its ancestral functional constraints, and enabled it to diversify into its current roles.

There is another huge and exciting question that needs answering. Archaea isn't the only taxonomic group lacking Hbs1 - shock horror - some Apicomplexan protists also don't carry it! So what's the mechanism in those guys? Has eukaryotic EF1A reacquired the ability to deliver eRF1 and Dom34p? That would be another amazing story!

Atkinson GC, Baldauf SL, & Hauryliuk V (2008). Evolution of nonstop, no-go and nonsense-mediated mRNA decay and their termination factor-derived components. BMC evolutionary biology, 8 PMID: 18947425

Evolution of Elongation Factor G

I got some good news the other day that my paper Evolution of elongation factor G and the origins of mitochondrial and chloroplast forms" has been accepted for publication in Molecular Biology and Evolution. Today it was published in an advance access format.
 Elongation factor G (EF-G) is a bacterial GTPase translation factor that binds to the ribosome and catalyses the movement of aminoacyl-tRNA from the A site to the P site (translocation). It also has a second function in ribosome recycling, where it interacts with RRF, the recycling factor, splitting the ribosomal subunits. EF-G is one of the most ancient proteins, with orthologues in archaea and eukaryotes (EF2), and it's also found in the eukaryotic organelles chloroplasts and mitochondria. Our paper is concerned with the bacterial and organellar versions.

I started working on EF-G during the early stages of my PhD in York with Sandra Baldauf. I found that there were two versions of EF-G in mitochondria (mtEFG1 and mtEFG2), and that these grouped with an odd selection of bacteria (spirochetes, planctomycetes and delta-proteobacteria, hence the name spdEFG1 and spdEFG2). The association of mitochondrial and spirochete EF-G had been noted briefly in Ford Doolittle's "Phylogenetic Classification and the Universal Tree" paper in Science , but there were no in depth analyses published. I presented my work at the SMBE meeting in Halifax, Nova Scotia in 2007, and we got the paper into shape for a manuscript. However, for several reasons (mainly that I was running out of time to submit my PhD and I needed to finish off my other thesis chapters), things got delayed and the paper was put on the shelf for a while. I came back to it when I started my post doc in 2008. In the meantime, the two paralogues were identified in yeast and spirochetes and biochemical investigations were carried out. This confirmed what we suspected: the two versions had become subfunctionalised for two facets of EF-G function, translocation and ribosome recycling. It was definitely high time to look at this in detail from an evolutionary perspective.

It's protein functional and structural evolution that I find most fascinating, so for me, the most exciting thing in our results is how asymmetric the subfunctionalisation has been. mt/spdEFG1 is very highly conserved, often with residues very different to classical EF-G (including a three amino acid insertion in the all-important Switch I region of the GTPase domain), whereas mt/spdEFG2 is much less conserved, even in the sites that are supposedly involved in its ribosome recycling function. It seems that for this function, all that's required is something roughly EF-G-shaped. Indeed, the most well conserved sites of spdEFG2 seem to be those essential for structural integrity. Just having this shadow, shell of an EF-G around to do the ribosome recycling seems to have freed spdEFG1 from some constraints and enabled it to become more highly specialised for its role in translocation.

So where to go from here with EF-G? Well we found that some delta-proteobacteria have all three types of EF-G (canonical EF-G, spdEFG1 and spdEFG2). I'm hoping that one day someone will check out these versions in depth in terms of recycling/translocation functionality and GTP hydrolysis activity. Maybe then we'll be be able to answer the burning question: just what does that Switch I insertion do?!

Atkinson GC, & Baldauf SL (2010). Evolution of elongation factor G and the origins of mitochondrial and chloroplast forms. Molecular biology and evolution PMID: 21097998

Doolittle, W. (1999). Phylogenetic Classification and the Universal Tree Science, 284 (5423), 2124-2128 DOI: 10.1126/science.284.5423.2124

Blog zero

Hello! Welcome to the blog!

I've decided that I should start blogging about my life and research. This is just a short post to sort of get the ball rolling. I imagine it will be an odd mix of sciencey stuff that I'm interested in, and some stories of life in Estonia.

I'm a post doc researcher at the University of Tartu. Some info about me and my research here. I moved to Tartu a couple of months ago from Sweden (I did my first post doc in Uppsala University) to join my partner Vasili Hauryliuk, who's been working part here and part in Uppsala for the last few years.

More info later!