How did UGA codon translation as tryptophan evolve in certain ciliates? A critique of Kachale et al. 2023 Nature

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition[1][1]. All non-standard ciliate genetic codes involve stop codon reassignments[1][1],[2][2],[3][3]. Two of these codes are ambiguous[1][1]–[3][3], with “stop” codons either translated or terminating translation depending on their context[2][2],[3][3]. Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia [4][4] and an alveolate species from the genus Amoebophrya [5][5]. Two ambiguous genetic codes in ciliates share translation of UGA “stop” codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons[1][1],[2][2]. Furthermore, though a UGA-cognate tRNACysUCA was reported in Euplotes [6][6], a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNATrpUCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNATrpCCA’s, rather than the typical 5 base pair stem tRNAs[7][7]. New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination[7][7]. Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing “stop” codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #ref-6 [7]: #ref-7