We have demonstrated here that tRNA misacylation with methionine occurs in S. cerevisiae
. This represents the second example of tRNA mismethionylation in cells in addition to mouse and humans. Since yeast is evolutionarily distant from mammals, this result suggests that tRNA mismethionylation is conserved from fungi to mammalian lineages. We also show that tRNA mismethionylation is derived from the activity of the MetRS. Mismethionylation occurs in vitro
when the yeast MetRS is associated with the other two proteins in the AME complex. This association in the complex, however, is not required for mismethionylation in vivo
as an Arc1
deletion yeast strain also shows similar level of mismethionylation. We further show by in vivo
utilization kinetics and proteomic mass spec analysis that misacylated tRNAs are used in translation in yeast, as in mammals (4
Our results lead to two wide-open biological questions. First, how do ribosome choose mismethionylated tRNAs in translation? Misacylated tRNA may or may not be used in translation depending on elongation factor selection and ribosome utilization. In E. coli
, misacylated tRNAs can bind to the elongation factor EF-Tu at different affinities compared to correctly charged tRNAs (30
). This differential binding has been shown to result in the exclusion of some misacylated tRNAs to EF-Tu binding while some other misacylated tRNAs cannot be properly delivered into the A-site of the ribosome (31
). How mismethionylated tRNAs are selected by EF-Tu is, however, unclear as the EF-Tu selection of misacylated tRNAs depends on the tRNA and the amino acid identity; mismethionylated tRNAs were not used in previous studies. In the fungal CTG clade species, the tRNA with anticodon CAG is charged with either serine or leucine, and both Ser and Leu charged tRNAs are used in translation (36
). This is one example of experimental evidence for the utilization of mischarged tRNAs in eukaryotes, suggesting that EF-1α does not vigorously discriminate misacylated tRNAs. Many studies have been conducted on how ribosome discriminates codon-anticodon mismatched tRNAs in the A and the P sites. Mismethionylated tRNAs, however, can enter the A site while maintaining perfect codon–anticodon matches. For instance, a mismethionylated tRNALysCUU
is expected to enter the A site containing the cognate AAG codon. We do not know how mismethionylated tRNA in the A site might perturb peptide bond formation or translocation, due to a lack of previous studies that specifically considers mismethionylated tRNAs.
Although EF-1α may not discriminate misacylated tRNAs, our proteomic analysis here shows that misacylated lysyl- and argininyl-tRNAs are used in translation. To identify the rules of ribosome utilization of mismethionylated tRNAs in the future, it should be possible to conduct proteomic studies to specifically identify Met substitutions in proteins that can be considered to derive from all the mismethionylated tRNA species. Each mismethionylated tRNA species is only present at an average level of ~0.5% of tRNAMets. If we consider that ribosome uses all such tRNAs, it will still represent a sub-1% presence of Met at individual non-Met positions in proteins. Met-containing peptides are also prone to oxidation in mass spectrometry analysis, which pose an additional challenge for proteome-wide identification. Unlike Cys-containing peptides, which can be specifically enriched from the proteomic mixture, no reliable chemical method is yet available to enrich Met-containing peptides.
The second biological question deals with the potential function of mis-translation using mismethionylated tRNAs. We have proposed previously for mammalian cells that low-frequency substitution of non-Met residues with Met in stress response proteins can enhance the known, protective function of genetically encoded Met residues against ROS inactivation of cells’ own proteins. ROS refers to a collection of highly reactive radicals or peroxides, byproducts of the electron transport chain, and as such used as signaling molecules for cell health and stress (37
). In mammals, ROS is also used as chemical weapons against invading microbes or undesired molecules. Their high chemical reactivity easily leads to damages of a cell’s own molecules, including proteins. To protect their own proteins against ROS inactivation, certain Met residues in an endogenous protein are positioned at strategic places to react first with diffusing ROS molecules before they can oxidize sensitive amino acid side chains in, e.g. an active site of an enzyme to result in permanent inactivation (5
). Our previous proposal suggests that substituting certain non-Met residues with Met during translation can enhance this effect, in particular, during oxidative stress response. Our yeast result here is consistent with this idea in that many tRNA encoding charged and polar amino acids are mismethionylated so that a Met substitution at these residues would enable such a protective function but less likely produce misfolded proteins. In order to test this functional proposal for mis-translation using mismethionylated tRNAs, one would need MetRS mutants that have diminished ability to mismethionylate but still maintain a wild-type level activity to charge tRNAMet
. Such MetRS mutants are available for the E. coli
), and we are actively identifying such mutants using the recombinant AME as the starting point.
To rule out misacylation caused by other enzymes in yeast cells, we purified the recombinant AME complex from E. coli and performed in vitro aminoacylation reactions with purified total yeast tRNA and tRNA transcripts. Mismethionylation was prevalent for many tRNAs and for all tRNAGlu transcripts tested. All the misacylated tRNAs in vitro are also misacylated in vivo, suggesting that the AME complex binds many tRNAs and facilitates interaction of many tRNAs with the MetRS for methionylation. It appears that the Arc1p protein in the AME complex facilitates interactions of MetRS with tRNAMet and a large number of other tRNAs. However, Arc1p is not required for methionylation in vivo.
The E. coli
MetRS misacylates only two E. coli
). These two tRNAs have anticodons that differ from the anticodon for methionine, CAU, by one nucleotide. The anticodon and several regions of the E. coli
MetRS that interact with the anticodon loop are responsible for misacylation. In addition, several studies have shown that the CAU anticodon plays a key role in yeast tRNA methionylation and discrimination (39
). As there are a large number of different misacylated tRNAs in S. cerevisiae
, the misacylated tRNAs have little similarity among their anticodons. This raised the question of how so many different tRNAs are misacylated with methionine in S. cerevisiae
. We found here that many tRNA species bind to the AME complex, including all the tRNAs that are misacylated. Since Arc1p is a generic tRNA-binding protein, Arc1p can bind many tRNAs and likely transfer them to the MetRS for aminoacylation with methionine, potentially explaining why the AME complex misacylates many tRNA species in vitro
In summary, tRNA mismethionylation occurs in mammals and as shown here in yeast. As in mammals, a large number of tRNAs can be mismethionylated in yeast, and these misacylated tRNAs are used in translation. The misacylated tRNAs code for charged or polar amino acids, corroborating a role for Met substituting more solvent exposed positions in proteins. This work provides another role of multisynthetase complexes in eukaryotes and expands our knowledge on the mystery of molecular and biological roles of mis-translation.