Identification of a non-canonical ciliate nuclear genetic code where UAA and UAG code for different amino acids

Abstract

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel uncultured ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3'UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

Fig 1. Genetic code change in Oligohymenophorea sp. PL0344.

Example multiple sequence alignment of a tubulin gamma chain protein and orthologous sequences spanning Eukaryota. The alignment has been trimmed for visualisation purposes to remove poorly conserved regions and highlight internal UAA and UAG codons.

Fig 2. Genetic code prediction for Oligohymenophorea sp. PL0344.

PhyloFisher genetic code prediction for the (A) UAA and (B) UAG codons using the PhyloFisher database of 240 orthologs. Only well conserved (>70%) amino acids are considered. Colours correspond to amino acid properties and match the multiple sequence alignment in Fig 1. (C) Codetta genetic code prediction. Log decoding probabilities for the UAA and UAG codons are shown for each of the 20 standard amino acids.

Fig 3. Example tRNA Genes.

(A) Predicted secondary structure of an example tRNA-Sup(UUA) predicted to function as a lysine tRNA. The wobble position is highlighted. According to wobble-binding rules, uracil at this position can bind to either adenine or guanine in the third codon position of mRNA, allowing the suppressor tRNA to recognise both UAA and UAG stop codons. (B) Predicted secondary structure of an example tRNA-Sup(CUA) predicted to function as a glutamic acid tRNA. (C) Predicted secondary structure of the tRNA-SeC(UCA) for selenocysteine.

Fig 4. Enrichment of tandem stop codons.

The proportion of codon positions occupied by UGA in the 20 in-frame codon positions immediately downstream of all genes and highly expressed genes. Positions where UGA is significantly overrepresented (chi-squared test, p-value < 0.05) are indicated with an asterisk.

Fig 5. Phylogenomic analysis of genetic code changes in the Ciliophora.

Maximum-likelihood phylogeny of 46 ciliate species and 9 outgroup species from the Alveolata, based on a concatenated alignment of 89 BUSCO proteins (40,289 amino acid sites) under the LG+F+I+R7 model using IQ-TREE. The values at branches represent statistical support from 100 non-parametric bootstraps with the LG+F+I+R7 model, 100 non-parametric bootstraps from the IQ-TREE partitioned analysis, and Bayesian posterior probabilities determined under the CAT-GTR model in PhyloBayes-MPI. Branches have full support from all three approaches (i.e., 100/100/1) except where indicated. Hyphens indicate branches that weren’t recovered under a particular analysis. Stop codon reassignments are shown (*, STOP; Q, glutamine; W, tryptophan; K, lysine; E, glutamic acid; Y, tyrosine; C, cysteine). Numbers inside solid black circles along branches indicate when a genetic code change event was inferred to have occurred (UAR = UAA and UAG). The percentage of proteins included in the concatenated alignment is shown in the bar plot, highlighting the amount of missing data per species.