CRACKING THE GENETIC CODE: UAA AND UAG UNIQUELY ENCODE AMINO ACIDS IN CILIATES

 

The genetic code combines three nucleotides to form codons specific for an amino acid or stop signal. There are 61 codons for amino acid (including the translation start codon AUG) synthesis and 3 codons as stop codons comprising 64 codons (UAA, UAG, and UGA). The concept of codon was proposed by Francis Crick and his colleagues in 1961, and only a few deviations have been observed over the “universal” genetic code. One of which is the codon UAA and UAG virtually have the same translation (i.e.) if there is a change in the translation of one codon, it is paired with the same change in the other which indicates that their evolution is coupled. There are rare variations from the canonical genetic code that are reported in various lineages,  including bacteria, viruses, and eukaryotic organisms with both organellar and nuclear genomes.

Ciliate, a group of single-celled eukaryotes (protists) from the phylum Ciliophora are a particular hotspot for genetic code variations. They are diverse because they contain two types of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), which contain their own distinct genome structure and function. The MIC genome is typically diploid and functions as a germline genome which is exchanged during sexual reproduction. The MIC genome undergoes rearrangement and excision during sexual reproduction and acts as a template for the generation of MAC which functions as a somatic genome that is typically short, fragmented, and gene-dense chromosome. 

There are so many significant changes in the stop codon with respect to the known genetic code of Ciliate. Some of the reported changes are UAA and UAG reassigned for glutamine in Tetrahymena, Paramecium, and Oxytricha, or glutamic acid in Campanella umbellaria and Carchesium polypinum or tyrosine in Mesodinium species. Other changes include coding tryptophan for UAG stop codon in Blepharisma or cysteine in Euplotes. Another remodelling genetic code found in Condylostoma magnum where the stop codon UAA, UAG, and UGA all code for amino acids (glutamine for UAA and UAG, and tryptophan for UGA). But some ciliates use canonical genetic codes like for example Fabrea salina, Litonotus pictus, and Stentor coeruleus. Note that in every case UAA and UAG code for the same amino acid.

Ciliate

Eukaryotes contain eukaryotic release factor (eRF1) which recognises the three stop codons in mRNA and triggers translation termination, with studies it has been shown that mutation in the N-terminus of eRF1 can alter the stop codon. Tandem stop codons are additional specific nucleotide stop codons presented in the 3’-untranslated region (3’-UTR) of the mRNA. They act as a backup stop codon providing an additional layer of protection over the translation process. They are present more than required in the case of ciliates compared to eukaryotes that use canonical genetic code. 

Several models have been proposed to understand the changes in the genetic code. The “Codon capture” model hypothesises that if the codon is rarely used then it is gradually eliminated and the corresponding tRNA also gets lost. However due to random genetic drift, the codon may appear again, but this time it may bind with the non-canonical tRNA which codes for another amino acid which results in a change of genetic code. In the “Ambiguous intermediate” model, it is proposed that the changes in the codon take place through an intermediate stage where the codon is ambiguously translated which can be recognized by multiple tRNAs that code for different amino acids. The process is driven by selection, and it results in the elimination of the cognate tRNA if the new meaning of the codon is advantageous. The “Genome streamlining” model says that in the case of organisms with small genomes like organellar genomes or parasites, due to the pressure to minimize the translational machinery and conserve space the genome code is or may alter. A recent model of the “tRNA Loss-Driven Codon Reassignment Model” states that codon reassignment can happen when there is a loss of tRNA or mutations in the release factor. This loss or alteration creates an unassigned codon. This unassigned codon can then be captured by another tRNA gene, leading to a change in the genetic code. 

In all genetic code changes reported till now, the codon UAA and UAG have the same amino acid, they are used as canonical stop codons or reassigned to the same amino acid. However novel variants in this codon have been found in ciliate belonging to the Oligohymenophorea class. Here the UAA and UAG codons have been reassigned to specific different amino acids. Researchers conducted a study using G&T-Seq (Genome and Transcriptome Sequencing), a sequencing technique that simultaneously analyzes both the genome and transcriptome of small groups of ciliate cells. By combining data from multiple samples, they were able to create a highly comprehensive macronuclear genome assembly and provide annotations. The analysis of the genome and transcriptome revealed that the UAA codon has been redefined to represent lysine, while the UAG codon now signifies glutamic acid. They identified several suppressor tRNA genes in the genome that support these changes in the genetic code. Notably, they found a significant presence of UGA codons in the 3'-untranslated regions (3'-UTR) of genes, suggesting that there is selective pressure to maintain tandem stop codons. This may serve a role in preventing errors in protein elongation in cases of translational readthrough. 

According to researchers, this is the first documented instance of a genetic code variant in which UAA and UAG codons have different meanings. This shows that in spite of the significant progress made in genomics over the past thirty years, there remain undiscovered irregularities in the genetic code that are yet to be uncovered in the natural world.

Reference:

McGowan J, Kilias ES, Alacid E, Lipscombe J, Jenkins BH, Gharbi K, et al. Identification of a non-canonical ciliate nuclear genetic code where UAA and UAG code for different amino acids. PLOS Genetics. 2023;19(10). doi:10.1371/journal.pgen.1010913