Earlier this year I was afforded the opportunity to hear Paul Schimmel, of the Scripps Research Institute, lecture on aminoacyl transfer RNA (tRNA) synthetases (aaRSs), a topic on which he is a leading authority. These enzymes attach particular amino acids to specific tRNA molecules for incorporating those amino acids into growing polypeptide chains by ribosomes. The basic features of these enzymes that contribute to the fundamental function of translating messenger RNAs (mRNAs) are conserved from humans through the most evolutionarily primitive single-celled organisms. Thus these enzymes might be presumed to correspond to prototypical vegetative gene products, i.e., gene products necessary for essential cellular functions that have little to do with more recently evolved functions beyond protein synthesis. So, it was extremely interesting to learn that in organisms that arose later in evolution there are other functions, unrelated to aminoacylation of tRNAs, associated with many of the aaRSs. These functions of aaRSs are summarized by Guo and Schimmel (Nature Chem. Biol., 2013).
Non-standard functions of highly conserved domains: A key feature of aaRS is that they possess binding sites for the specific amino acids that they covalently attach to the particular tRNA molecules that collaborate with the ribosome and messenger RNA (mRNA) to incorporate amino acids into growing polypeptide chains as directed by the sequence of codons in the mRNAs. These amino acid-specific binding sites that are essential for the canonical function of aaRS molecules in attaching those amino acids to tRNA molecules, lend themselves to exploitation for other cellular purposes of several sorts. Below are three examples:
LeuRS: In both yeast and mammalian cells, the leucine tRNA aminoacyl synthetase (LeuRS) serves as a sensor for free leucine. When LeuRS binds leucine in human cells, it interacts with a signaling complex of multiple proteins known as mTORC1 and activates this complex. If mTORC1 is inactive, as is the case when cells are starved for amino acids, translation of mRNAs is halted at an early step. A domain of LeuRS that is found in higher eukaryotes but not more evolutionarily primitive species is involved in the direct interaction with mTORC1 and is presumed to be activated by the binding of leucine, to a highly conserved domain (catalytic module), through inter-domain transmission of a conformational change.
GlnRS: Binding of glutamine to GlnRS decreases the activity of apoptosis signal-regulating kinase 1 (ASK1) that leads to apoptotic cell death. Consistent with the preceding claim, limiting access to glutamine can activate ASK1 and lead to apoptosis in a human embryonic kidney cell line, HEK293 cells.
LysRS: This synthetase, along with a few other tRNA synthetases, can generate the metabolite diadenosine tetraphosphate (Ap4A). When mast cells are activated by crosslinking of the Fcepsilon receptor I (FcepsilonRI) that binds free IgE molecules, they activate the mitogen-activated protein kinase signaling pathway, which phosphorylates LysRS. Once phosphorylated, LysRS translocates to the nucleaus and increases its production of Ap4A. The increased amounts of Ap4A activates the function of microphthalmia-associated transcription factor (MITF) leading to transcription and translation of genes that contribute to immune mechanisms.
Non-standard functions of “newer” domains: Additional nontranslational functions of aaRSs are associated with domains that were added at different stages of phylogenetic evolution. Guo and Schimmel suggest that the progressive addition these “new” domains to aaRS is not observed with other phylogenetically old protein families. They also note that domain additions are often seen at points in evolution at which major transitions occurred. For example, the WHEP domain, so-named because of the single letter abbreviations for the amino acids of the corresponding aaRSs [W=tryptophan(Trp)RS; H=histidine(His)RS; EP= glutamic acid/proline(GluPro)RS], is first observed in the insects but retained in species arising later in evolution.
While deletion of the WHEP domain of human TrpRS has minimal impact on the aminoacylation performed by this synthetase, this portion of the protein has been found to mediate interactions with molecules that are involved in activating a key regulator of cellular proliferation, p53. These effects of the TrpRS molecule are increased by the cytokine interferon-gamma (IFNg) and appear to mediate the ability of IFNg to inhibit the proliferation of cells that display the IFNg receptor.
There are other examples of cellular functions other than aminoacylation of tRNAs that phylogenetically newer aaRS domains serve:
1) GluProRS WHEP domains – IFNg-mediated inhibition of translation of mRNAs;
2) SerRS UNE-S (unique to SerRS) domain – nuclear localization of SerRS and transcriptional regulation of genes encoding products involved in the vascular endothelial growth factor A signaling pathway thereby influencing the development of a closed circulatory system; the SerRS UNE-S domain is not needed for aminoacylation activity.
Non-translational functions of aaRS-associated proteins: Three proteins that interact in a large multi-protein complex, termed the MSC, involving nine different aaRSs have been demonstrated to perform functions unrelated to translation. For example, one of these MSC proteins when free of the complex can act as a cytokine that inhibits cellular proliferation and favors apoptosis. A second of the MSC proteins when free of the complex is involved in mediating the response to DNA damage.
Non-translational functions of aaRS-derived fragments: Portions of aminoacyl tRNA-synthetases can be generated by alternative splicing of the corresponding mRNAs or by proteolytic cleavage. Examples of such structure-function associations are:
1) TyrRS – proteolysis of secreted TyrRS can produce N- and C-terminal fragments that respectively elicit a) neutrophil migration into a tissue and b) promote inflammation while inhibiting angiogenesis;
2) TrpRS – a splice variant effectively inhibits angiogenesis;
3) GluProRS – a proteolytic fragment inhibits translation of mRNAs encoding gene products that contribute to inflammation.
Clinical correlations of aaRSs: Accumulating evidence suggests that aaRS molecules are relevant to both disease pathogenesis and potentially even therapy. For example, there are mutations in human aaRSs that are associated with clinical conditions. Mutations in four aaRS molecules (AlaRS, GlyRS, LysRS, and TyrRs) are associated with the heritable peripheral neuropathy, Charcot-Marie-Tooth disease. There is evidence to suggest that at least some of the relevant mutations contribute to the neuropathy via mechanisms not directly related to effects on aminoacylation of tRNAs. Other aaRSs may be useful as biomarkers in human diseases or as therapeutic agents.
The major take-home message is that even though aaRS genes and proteins are as central to basic cellular functions as almost any proteins, they have been impressively integrated into an amazing variety of cellular mechanisms and pathways not directly related to their canonical functions in aminoacylation of tRNAs. Such diverse structure-function correlations point to the general inadequacy of distinctions between molecules that mediate so-called “vegetative” or “housekeeping” functions and those that mediate what have been called “contingency” functions (Greenspan, 1998). They also highlight the essential fluidity of genes and genomes, as noted by Graur et al. (Genome Biol Evol., 2015) in the context of offering an evolutionarily-based functional classification of genes. These latter authors note that any genomic sequence classified in one functional category at a given time can with a finite probability be transformed by a mutational event into a sequence that belongs in a different functional category. So, in considering aaRSs, as we have seen in many other contexts, evolution can pursue whatever “creative” and quasi-random opportunities to enhance fitness present themselves and violate any of our pre-conceived notions of how cells and molecules should behave, what I have referred to as the Principle of Radical Evolutionary Indifference.
Guo M, Schimmel P. Essential nontranslational functions of tRNA synthetases. Nat Chem Biol. 2013 Mar;9(3):145-53. doi: 10.1038/nchembio.1158. Review. PubMed PMID: 23416400; PubMed Central PMCID: PMC3773598.
Greenspan NS. Genomic logic, allelic inference, and the functional classification of genes. Perspect Biol Med. 1998 Spring;41(3):409-16.
Graur D, Zheng Y, Azevedo RB. An Evolutionary Classification of Genomic Function. Genome Biol Evol. 2015 Jan 28;7(3):642-645.