RQC is a critical pathway conserved across animals, essentially acting as the cell’s last resort for the rescue of “jammed” ribosomes. A jammed ribosome is one that stalls on the mRNA during translation, preventing further reading along the mRNA and the release of the elongating peptide chain. Disruptions to this pathway have been associated with several ribosomopathies, or human disease conditions related to ribosome malfunction.
Since the RQC pathway was first discovered around fifteen years ago, its steps have been more or less worked out: very generally, the jammed ribosome is split into its two subunits, the faulty mRNA is removed and degraded, the incomplete peptide chain is elongated and pushed out through the exit tunnel via a remarkable tailing process [
Shen et al.], and the chain itself is concomitantly tagged for degradation. Still, one outstanding question remained: across life, elongating peptide chains can only be released from the ribosome by the action of a release factor, which severs the last amino acid residue from the tRNA to which it is bound. In RQC, the responsible release factor was unknown.
Our group has had a longstanding interest in the evolution of both release factors and the RQC pathway [
Leipe et al.][
Burroughs and Aravind]. Through a collaborative effort with the
Deshaies lab, we discovered the elusive RQC release factor. In humans, it is found in the ANKZF1 protein; its yeast ortholog is Vms1. Several elegant experiments clearly demonstrating the tRNA-aminoacyl hydrolase activity of Vms1/ANKZF1 and its impact on the degradation of the stalled peptide chain were recently published in the Nature magazine [
Verma et al.]. Given that the ANKZF1 protein was recently directly linked to infantile-onset inflammatory bowel disease [
van Haaften-Visser et al.], this study both links the condition to ribosome dysfunction and provides the first genetic function leads for a serious contributor to this complex condition.
While this discovery finally completes our understanding of the central steps in the RQC pathway, it also throws new light on another long-standing mystery: the deep evolution of release factor proteins. While most core pieces of the translation machinery are universally-conserved across all Life, the solution to the translation termination step differs in both the archeao-eukaryotic and bacterial lineages, which each have their own distinct release factors. It is possible that one of these release factors was present in the last universal common ancestor (LUCA) and was displaced in the other lineage, or both lineages independently-evolved their own solutions.
We found that the Vms1/ANKZF1 release factor was part of a previously-unknown and extensive radiation of release factors related to the archaeo-eukaryotic release factors (aeRF1s). Surprisingly, these new members were also found across a broad range of bacteria; the first discovery of any aeRF1s in bacteria. Careful analysis of these new versions allowed us to divide them into two groups, what we call the VLRF1 clade (for Vms1/ANKZF1-like release factor clade) and the baeRF1 clade (for bacterial archaeo-eukaryotic release factor clade). We predict that the release factor proteins belonging to the first clade, including those found in the bacteria, are all likely to be involved in the rescuing of jammed ribosomes. One of the more striking evolutionary observations of this clade is that the direct antecedents of the eukaryote Vms1/ANKZF1 release factor proteins are found almost exclusively in the Bacteroidetes lineage of the bacteria. While the bulk of the bacterially-inherited genetic component of the eukaryotes was inherited from the a-proteobacterial mitochondrial symbiont progenitor, this finding adds to a growing list of key eukaryotic protein domains which have their roots in early symbionts from other lineages.
The second group, the baeRF1 clade, is found only in diverse bacteria species. These release factors are notably under tremendous selective pressure: in other words, they are evolving rapidly in terms of their structure and sequence features. In fact, we predict that most the versions belonging to this clade are likely to be catalytically inactive, incapable of cleaving tRNA-aminoacyl bonds in the ribosome. This begs the question: what is their function in these bacteria, and why are they evolving so rapidly? Our analysis identified a conserved link in bacterial between baeRF1-like proteins and ribosome hibernation factors (Figure 1). We suspect that baeRF1 proteins are likely involved in bacterial conflict: potentially activated in response to virus or other invasive element infection and then contributing to either the shutting down or re-starting of translation along with the ribosome hibernation factor.
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| Figure 1. Gene neighborhoods of baeRF1 |
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While our findings speak to a previously poorly-understood complexity in the evolution of aeRF1 proteins, ultimately little is revealed about the state of the release factor in the LUCA. While it might be tempting to speculate that the VLRF1 and baeRF1 clades represent surviving remnants of a potential ancestral bacterial aeRF1 presence displaced early in bacteria by the bacteria-specific release factor fold, our analysis indicates that both clades likely emerged from later transfers from a classical archaeal aeRF1 progenitor.
