Comparative-genomic analysis indicates that most archaea encode members of two major groups of Ubl proteins with the β
-grasp fold, the ThiS and MoaD families. The ThiS family genes are rarely found together with genes for thiamine and Mo/W cofactor metabolism enzymes but instead are often associated with various highly conserved and probably essential genes with functions related to translation, especially, tRNA modification. Thus, most if not all ThiS family proteins are predicted to function as sulfur carrier proteins for reactions similar to those recently characterized for the URM1 pathway in yeast [37
]. In contrast, genomic associations suggest that the primary function of the MoaD family proteins is indeed the Mo/W cofactor biosynthesis. The absence of Ubl proteins and E1-like Ubl-activating enzymes of the arCOG1676 in such autotrophic archaea as M. jannaschii
and M. kandleri
and the absence of association of Ubl genes with thiamine biosynthesis genes (other than ThiI family enzymes which are probably involved in tRNA modification) is compatible with the existence of an alternative thiamine biosynthesis pathway in archaea.
Surprisingly, despite their apparent functional preferences, ThiS and MoaD family members appear to be interchangeable in pathways that employ Ubl proteins either as sulfur carriers or for protein modification. This possibility is born out both through analysis of gene associations for both subfamilies as described here and by the experimental data on the two SAMP proteins of Haloferax volcanii
one of which belongs to the ThiS family and the other one to the MoaD family [21
The most prominent associations revealed by comparative genomics for the archaeal Ubl proteins are with enzymes of tRNA modification. This finding leads to the hypothesis that the majority of the β
-grasp Ubl proteins in archaea, at least those of the ThiS family, are involved in sulfur insertion steps of the biosynthesis of modified nucleotides. Given the ubiquity of a variety of tRNA modifications across cellular life [63
], this is likely to be the ancestral function of the Ubl proteins that subsequently were recruited for other chemically similar reactions, such as MoCo and thiamine biosynthesis, as well as protein modification. This hypothesis is compatible with the role of the eukaryotic Urm1 protein in specific tRNA modification and with fusion of the Ubl domain to the KEOPS complex subunit Cgi121, given the requirement of KEOPS for the t6A modification. Experimental study of the involvement of Ubl proteins in tRNA modification appears to be an extremely promising research direction.
From a more general perspective, tRNA modification is undoubtedly a major mechanism of the quality control of translation [64
]. Considering also the association of another KEOPS subunit (Pcc1) with the exosome and the proteasome, it is tempting to view the Ubl proteins as general devices for protein quality control, both at the most fundamental level of translation fidelity and at the secondary levels of regulated protein and RNA degradation. In eukaryotes, the latter mechanisms assumed hugely diversified roles which required the evolution of the enormously complex Ub-centered signaling systems.
The comparative-genomic analysis of the genes for Ubl proteins and the enzymes that appear functionally linked to them suggests that archaea might possess still uncharacterized Ubl-related functional systems. In particular, the association of the Jab protease with a cytidylyltransferase-like enzyme appears to be a candidate for a Ubl conjugation/deubiquitination system. In addition, archaea are likely to possess functional analogs of Ubl proteins that are structurally and hence evolutionarily unrelated to the β-grasp fold. This group includes small proteins of the TBP-like fold that bend at a GG doublet and are often fused to E1 family enzymes, in a strong indication of their Ubl-type activity, along with putative homologs of the bacterial Pup protein.
In conclusion, the comparative-genomic analysis triggered by the seminal discovery of the SAMPylation reactions in H. volcanii reveals unexpected potential complexity of archaeal Ubl-centered systems and offers several directions for further experimentation, the most important of which arguably is the validation of the hypothesis on the involvement of Ubl proteins in tRNA modification. In addition, this analysis opens up an unexpected and potentially fundamental area of inquiry into the evolution of cells, namely, the ancestral connection between systems of protein quality control that operate at different levels.