An unprecedented codon reassignment and two new genetic code variants in ciliates

Ciliates are well-known for frequent nuclear genetic code changes. However, the exploration of ciliate nuclear genomes is an unfinished enterprise, and particular genetic code variants have been previously attributed to some ciliate lineages without solid evidence. We focused on two such groups, the class Litostomatea and deeply-branching lineages of the class Spirotrichea, and by analysing their genomes and transcriptomes we found that some of them employ genetic code variants differing from those suggested before. Particularly notable is the spirotrichean genus Licnophora, which we found to exhibit an unprecedented combination of codon reassignments, i.e. a new genetic code variant. In Licnophora spp., each of the three standard stop codons has a different altered meaning: UAA encodes glutamate, UAG glutamine, and UGA exhibits homonymy, encoding arginine when present in-frame and terminating translation at the coding sequence ends. Contrary to some previous claims, our analyses demonstrated that litostomateans generally employ the standard genetic code, with a single identified exception, the species Balantidium ctenopharyngodoni, which exhibits a codon reassignment never reported before: UGA encoding leucine. Whereas tRNAs decoding the reassigned UAA and UAG codons in Licnophora and UGA in B. ctenopharyngodoni were readily identified in the respective genomic data, the mechanism of UGA decoding as arginine in Licnophora and certain other spirotricheans remains elusive. Our results paint a more complex picture of genetic code evolution in Spirotrichea than assumed before and necessitate an update of the current list of genetic code variants to allow for an accurate prediction of protein sequences in ciliates.