Natural reassignment of CUU and CUA sense codons to alanine in Ashbya mitochondria

Abstract

The discovery of diverse codon reassignment events has demonstrated that the canonical genetic code is not universal. Studying coding reassignment at the molecular level is critical for understanding genetic code evolution, and provides clues to genetic code manipulation in synthetic biology. Here we report a novel reassignment event in the mitochondria of Ashbya (Eremothecium) gossypii, a filamentous-growing plant pathogen related to yeast (Saccharomycetaceae). Bioinformatics studies of conserved positions in mitochondrial DNA-encoded proteins suggest that CUU and CUA codons correspond to alanine in A. gossypii, instead of leucine in the standard code or threonine in yeast mitochondria. Reassignment of CUA to Ala was confirmed at the protein level by mass spectrometry. We further demonstrate that a predicted is transcribed and accurately processed in vivo, and is responsible for Ala reassignment. Enzymatic studies reveal that is efficiently recognized by A. gossypii mitochondrial alanyl-tRNA synthetase (AgAlaRS). AlaRS typically recognizes the G3:U70 base pair of tRNAAla; a G3A change in Ashbya abolishes its recognition by AgAlaRS. Conversely, an A3G mutation in Saccharomyces cerevisiae confers tRNA recognition by AgAlaRS. Our work highlights the dynamic feature of natural genetic codes in mitochondria, and the relative simplicity by which tRNA identity may be switched.

A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes

“…The second scenario is represented by the mitochondrial tRNA Ala UAG in Eremothecium, which originated from a tRNA Thr UAG by acquiring the acceptor stem identity determinant "G3:U70" (Supplemental Fig. S13; Ling et al 2014). This tRNA Ala UAG is charged by the AlaRS, which is nondiscriminative against the anticodon.…”

“…It is supposed that CUG-codon decoding is ambiguous in many extant Candida species Suzuki et al 1997), that the CUG-codon decoding can-at least in part-be converted Bezerra et al 2013), and that the origin of the tRNA which has evolved from a tRNA His ancestor (Su et al 2011). In the Eremothecium subbranch, the CUG codons are decoded by alanine, but this modified code did not originate by capture of the CUG codons by an anticodon-mutated tRNA Ala but by switching the acceptor stem identity determinants of the Saccharomycetaceae tRNA Thr from threonine to alanine (Ling et al 2014). From the analysis of the conservation of amino acid types and CUG-codon positions in motor and cytoskeletal proteins, we recently showed that these proteins allow us to unambiguously assign the code employed by any given species of yeast (Mühlhausen and Kollmar 2014).…”

Pathways of Genetic Code Evolution in Ancient and Modern Organisms

“…Such reassignments require only modifications in the anticodon of one tRNA (gain) together with the loss of functionality of the original tRNA. However, there exist a few examples of sense to sense codon reassignments, such as CUN:Leu to Thr and CUU, CUA: Thr to Ala (Osawa et al 1990;Su et al 2011;Ling et al 2014), where the reassigned amino acid is in a different column of the code relative to the original assignment. Such reassignments typically require tRNA duplication followed by modifications in the tRNA identity elements that lead to changes in charging specificity.…”

Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology

“…In another closely related species, Ashbya gossypii , a tRNA Ala UAG derived from either tRNA His GUG or tRNA Thr UAG reassigns mitochondrial CUA and CUU codons to Ala, and CUC and CUG codons are absent in mitochondrial genes 30 . This leaves UUG and UUA as the only Leu codons in the A. gossypii mitochondrial genome.…”