BACKGROUND: Departures from the standard genetic code in eukaryotic nuclear genomes are known for only a handful of lineages and only a few genetic code variants seem to exist outside the ciliates, the most creative group in this regard. Most frequent code modifications entail reassignment of the UAG and UAA codons, with evidence for at least 13 independent cases of a coordinated change in the meaning of both codons. However, no change affecting each of the two codons separately has been documented, suggesting the existence of underlying evolutionary or mechanistic constraints. RESULTS: Here, we present the discovery of two new variants of the nuclear genetic code, in which UAG is translated as an amino acid while UAA is kept as a termination codon (along with UGA). The first variant occurs in an organism noticed in a (meta)transcriptome from the heteropteran Lygus hesperus and demonstrated to be a novel insect-dwelling member of Rhizaria (specifically Sainouroidea). This first documented case of a rhizarian with a non-canonical genetic code employs UAG to encode leucine and represents an unprecedented change among nuclear codon reassignments. The second code variant was found in the recently described anaerobic flagellate Iotanema spirale (Metamonada: Fornicata). Analyses of transcriptomic data revealed that I. spirale uses UAG to encode glutamine, similarly to the most common variant of a non-canonical code known from several unrelated eukaryotic groups, including hexamitin diplomonads (also a lineage of fornicates). However, in these organisms, UAA also encodes glutamine, whereas it is the primary termination codon in I. spirale. Along with phylogenetic evidence for distant relationship of I. spirale and hexamitins, this indicates two independent genetic code changes in fornicates. CONCLUSIONS: Our study documents, for the first time, that evolutionary changes of the meaning of UAG and UAA codons in nuclear genomes can be decoupled and that the interpretation of the two codons by the cytoplasmic translation apparatus is mechanistically separable. The latter conclusion has interesting implications for possibilities of genetic code engineering in eukaryotes. We also present a newly developed generally applicable phylogeny-informed method for inferring the meaning of reassigned codons.

Canadian Institute for Advanced Research Program in Integrated Microbial Biodiversity Toronto ON Canada

Centre for Comparative Genomics and Evolutionary Bioinformatics Dalhousie University Halifax NS Canada

Department of Biochemistry and Molecular Biology Dalhousie University Halifax NS B3H 4R2 Canada

Department of Biology and Ecology Faculty of Science University of Ostrava Chittussiho 10 710 00 Ostrava Czech Republic

Department of Mathematics and Statistics Dalhousie University Halifax NS B3H 4R2 Canada

Department of Zoology Faculty of Science Charles University Viničná 7 128 00 Prague Czech Republic

Institute of Molecular Genetics Academy of Sciences of the Czech Republic Vídeňská 1083 142 20 Prague Czech Republic

Unité d'Ecologie Systématique et Evolution Centre National de la Recherche Scientifique Université Paris Sud Paris Saclay AgroParisTech Orsay France

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Knight RD, Freeland SJ, Landweber LF. Rewiring the keyboard: evolvability of the genetic code. Nat Rev Genet. 2001;2:49–58. doi: 10.1038/35047500. PubMed DOI

Ling J, O'Donoghue P, Söll D. Genetic code flexibility in microorganisms: novel mechanisms and impact on physiology. Nat Rev Microbiol. 2015;13:707–21. doi: 10.1038/nrmicro3568. PubMed DOI PMC

Keeling PJ. Evolution of the genetic code. Curr Biol. 2016;26:R851–3. doi: 10.1016/j.cub.2016.08.005. PubMed DOI

Matsumoto T, Ishikawa SA, Hashimoto T, Inagaki Y. A deviant genetic code in the green alga-derived plastid in the dinoflagellate Lepidodinium chlorophorum. Mol Phylogenet Evol. 2011;60:68–72. PubMed

Preer Jr JR, Preer LB, Rudman BM, Barnett AJ. Deviation from the universal code shown by the gene for surface protein 51A in Paramecium. Nature. 1985;314:188–90. PubMed

Horowitz S, Gorovsky MA. An unusual genetic code in nuclear genes of Tetrahymena. Proc Natl Acad Sci U S A. 1985;82:2452–5. PubMed PMC

Sánchez-Silva R, Villalobo E, Morin L, Torres A. A new noncanonical nuclear genetic code: translation of UAA into glutamate. Curr Biol. 2003;13:442–7. doi: 10.1016/S0960-9822(03)00126-X. PubMed DOI

Keeling PJ, Leander BS. Characterisation of a non-canonical genetic code in the oxymonad Streblomastix strix. J Mol Biol. 2003;326:1337–49. PubMed

Lozupone CA, Knight RD, Landweber LF. The molecular basis of nuclear genetic code change in ciliates. Curr Biol. 2001;11:65–74. doi: 10.1016/S0960-9822(01)00028-8. PubMed DOI

Santos MA, Ueda T, Watanabe K, Tuite MF. The non-standard genetic code of Candida spp.: an evolving genetic code or a novel mechanism for adaptation? Mol Microbiol. 1997;26:423–31. PubMed

de Koning AP, Noble GP, Heiss AA, Wong J, Keeling PJ. Environmental PCR survey to determine the distribution of a non-canonical genetic code in uncultivable oxymonads. Environ Microbiol. 2008;10:65–74. PubMed

Cocquyt E, Gile GH, Leliaert F, Verbruggen H, Keeling PJ, De Clerck O. Complex phylogenetic distribution of a non-canonical genetic code in green algae. BMC Evol Biol. 2010;10:327. doi: 10.1186/1471-2148-10-327. PubMed DOI PMC

Karpov SA, Mikhailov KV, Mirzaeva GS, Mirabdullaev IM, Mamkaeva KA, Titova NN, Aleoshin VV. Obligately phagotrophic aphelids turned out to branch with the earliest-diverging fungi. Protist. 2013;164:195–205. doi: 10.1016/j.protis.2012.08.001. PubMed DOI

Mühlhausen S, Findeisen P, Plessmann U, Urlaub H, Kollmar M. A novel nuclear genetic code alteration in yeasts and the evolution of codon reassignment in eukaryotes. Genome Res. 2016;26:945–55. doi: 10.1101/gr.200931.115. PubMed DOI PMC

Riley R, Haridas S, Wolfe KH, Lopes MR, Hittinger CT, Göker M, Salamov AA, Wisecaver JH, Long TM, Calvey CH, et al. Comparative genomics of biotechnologically important yeasts. Proc Natl Acad Sci U S A. 2016;113:9882–7. doi: 10.1073/pnas.1603941113. PubMed DOI PMC

Swart EC, Serra V, Petroni G, Nowacki M. Genetic codes with no dedicated stop codon: context-dependent translation termination. Cell. 2016;166:691–702. doi: 10.1016/j.cell.2016.06.020. PubMed DOI PMC

Heaphy SM, Mariotti M, Gladyshev VN, Atkins JF, Baranov PV. Novel ciliate genetic code variants including the reassignment of all three stop codonsto sense codons in Condylostoma magnum. Mol Biol Evol. 2016;33:2885–9. PubMed PMC

Záhonová K, Kostygov A, Ševčíková T, Yurchenko V, Eliáš M. An unprecedented non-canonical nuclear genetic code with all three termination codons reassigned as sense codons. Curr Biol. 2016;26:2364–9. doi: 10.1016/j.cub.2016.06.064. PubMed DOI

Suzuki T, Numata T. Convergent evolution of AUA decoding in bacteria and archaea. RNA Biol. 2014;11:1586–96. doi: 10.4161/15476286.2014.992281. PubMed DOI PMC

Hanyu N, Kuchino Y, Nishimura S, Beier H. Dramatic events in ciliate evolution: alteration of UAA and UAG termination codons to glutamine codons due to anticodon mutations in two Tetrahymena tRNAs. EMBO J. 1986;5:1307–11. PubMed PMC

Laforest MJ, Roewer I, Lang BF. Mitochondrial tRNAs in the lower fungus Spizellomyces punctatus: tRNA editing and UAG ‘Stop’ codons recognized as leucine. Nucleic Acids Res. 1997;25:626–32. PubMed PMC

Fučíková K, Lewis PO, González-Halphen D, Lewis LA. Gene arrangement convergence, diverse intron content, and genetic code modifications in mitochondrial genomes of Sphaeropleales (Chlorophyta) Genome Biol Evol. 2014;6:2170–80. doi: 10.1093/gbe/evu172. PubMed DOI PMC

Korostelev AA. Structural aspects of translation termination on the ribosome. RNA. 2011;17:1409–21. doi: 10.1261/rna.2733411. PubMed DOI PMC

Keeling PJ, Burki F, Wilcox HM, Allam B, Allen EE, Amaral-Zettler LA, Armbrust EV, Archibald JM, Bharti AK, Bell CJ, et al. The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol. 2014;12:e1001889. doi: 10.1371/journal.pbio.1001889. PubMed DOI PMC

Hull JJ, Geib SM, Fabrick JA, Brent CS. Sequencing and de novo assembly of the western tarnished plant bug (Lygus hesperus) transcriptome. PLoS One. 2014;8:e55105. PubMed PMC

Lapointe FJ, Lopez P, Boucher Y, Koenig J, Bapteste E. Clanistics: a multi-level perspective for harvesting unrooted gene trees. Trends Microbiol. 2010;18:341–7. doi: 10.1016/j.tim.2010.03.009. PubMed DOI

Bass D, Silberman JD, Brown MW, Pearce RA, Tice AK, Jousset A, Geisen S, Hartikainen H. Coprophilic amoebae and flagellates, including Guttulinopsis, Rosculus and Helkesimastix, characterise a divergent and diverse rhizarian radiation and contribute to a large diversity of faecal-associated protists. Environ Microbiol. 2016;18:1604–19. PubMed

Hayashi-Ishimaru Y, Ohama T, Kawatsu Y, Nakamura K, Osawa S. UAG is a sense codon in several chlorophycean mitochondria. Curr Genet. 1996;30:29–33. doi: 10.1007/s002940050096. PubMed DOI

Roth A, Anisimova M, Cannarozzi GM. Measuring codon usage bias. In: Cannarozzi GM, Schneider A, editors. Codon Evolution: Mechanisms and Models. New York: Oxford University Press; 2012. pp. 189–217.

Yubuki N, Zadrobílková E, Čepička I. Ultrastructure and molecular phylogeny of Iotanema spirale gen. et sp. nov., a new lineage of endobiotic Fornicata with strikingly simplified morphology and ultrastructure. J Euk Microbiol. 2016. doi:10.1111/jeu.12376. Ahead of print. PubMed

Kolisko M, Silberman JD, Cepicka I, Yubuki N, Takishita K, Yabuki A, Leander BS, Inouye I, Inagaki Y, Roger AJ, et al. A wide diversity of previously undetected free-living relatives of diplomonads isolated from marine/saline habitats. Environ Microbiol. 2010;12:2700–1270. PubMed

Takishita K, Kolisko M, Komatsuzaki H, Yabuki A, Inagaki Y, Cepicka I, Smejkalová P, Silberman JD, Hashimoto T, Roger AJ, et al. Multigene phylogenies of diverse Carpediemonas-like organisms identify the closestrelatives of ‘amitochondriate’ diplomonads and retortamonads. Protist. 2012;163:344–55. PubMed

NCBI Genetic Code Table. National Center for Biotechnology Information, Maryland, USA. 2006. ftp://ftp.ncbi.nih.gov/entrez/misc/data/gc.prt. Accessed 21 Aug 2016.

Graur D. Molecular and Genome Evolution. Sunderland: Sinauer Associates; 2016.

Elzanowski A, Ostell J. The Genetic Codes (last update 30 Apr 2013). National Center for Biotechnology Information, Maryland, USA. 2013. http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi. Accessed 21 Aug 2016.

Brown MW, Kolisko M, Silberman JD, Roger AJ. Aggregative multicellularity evolved independently in the eukaryotic supergroup Rhizaria. Curr Biol. 2012;22:1123–7. doi: 10.1016/j.cub.2012.04.021. PubMed DOI

Keeling PJ, Doolittle WF. A non-canonical genetic code in an early diverging eukaryotic lineage. EMBO J. 1996;15:2285–90. PubMed PMC

Kolisko M, Cepicka I, Hampl V, Leigh J, Roger AJ, Kulda J, Simpson AGB, Flegr J. Molecular phylogeny of diplomonads and enteromonads based on SSU rRNA, alpha-tubulin and HSP90 genes: Implications for the evolutionary history of the double karyomastigont of diplomonads. BMC Evol Biol. 2008;8:1. doi: 10.1186/1471-2148-8-205. PubMed DOI PMC

Breitschopf K, Achsel T, Busch K, Gross HJ. Identity elements of human tRNA(Leu): structural requirements for converting human tRNA(Ser) into a leucine acceptor in vitro. Nucleic Acids Res. 1995;23:3633–7. doi: 10.1093/nar/23.18.3633. PubMed DOI PMC

Giegé R, Sissler M, Florentz C. Universal rules and idiosyncratic features in tRNA identity. Nucleic Acids Res. 1998;26:5017–35. doi: 10.1093/nar/26.22.5017. PubMed DOI PMC

Pang YL, Poruri K, Martinis SA. tRNA synthetase: tRNA aminoacylation and beyond. Wiley Interdiscip Rev RNA. 2014;5:461–80. doi: 10.1002/wrna.1224. PubMed DOI PMC

Schüll C, Beier H. Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro. Nucleic Acids Res. 1994;22:1974–80. doi: 10.1093/nar/22.11.1974. PubMed DOI PMC

Nakamura Y, Ito K. How protein reads the stop codon and terminates translation. Genes Cells. 1998;3:265–78. doi: 10.1046/j.1365-2443.1998.00191.x. PubMed DOI

Bertram G, Bell HA, Ritchie DW, Fullerton G, Stansfield I. Terminating eukaryote translation: domain 1 of release factor eRF1 functions in stop codon recognition. RNA. 2000;6:1236–47. doi: 10.1017/S1355838200000777. PubMed DOI PMC

Blanchet S, Rowe M, Von der Haar T, Fabret C, Demais S, Howard MJ, Namy O. New insights into stop codon recognition by eRF1. Nucleic Acids Res. 2015;43:3298–308. doi: 10.1093/nar/gkv154. PubMed DOI PMC

Brown A, Shao S, Murray J, Hegde RS, Ramakrishnan V. Structural basis for stop codon recognition in eukaryotes. Nature. 2015;524:493–6. doi: 10.1038/nature14896. PubMed DOI PMC

Inagaki Y, Blouin C, Doolittle WF, Roger AJ. Convergence and constraint in eukaryotic release factor 1 (eRF1) domain 1: the evolution of stop codon specificity. Nucleic Acids Res. 2002;30:532–44. doi: 10.1093/nar/30.2.532. PubMed DOI PMC

Bezerra AR, Guimarães AR, Santos MA. Non-standard genetic codes define new concepts for protein engineering. Life (Basel) 2015;5:1610–28. PubMed PMC

Conard SE, Buckley J, Dang M, Bedwell GJ, Carter RL, Khass M, Bedwell DM. Identification of eRF1 residues that play critical and complementary roles in stop codon recognition. RNA. 2012;18:1210–21. doi: 10.1261/rna.031997.111. PubMed DOI PMC

Jacob JEM, Vanholme B, Van Leeuwen T, Gheysen G. A unique genetic code change in the mitochondrial genome of the parasitic nematode Radopholus similis. BMC Res Notes. 2009;2:192. PubMed PMC

Scott DR. An annotated listing of host plants of Lygus hesperus Knight. Entomol Soc Am Bull. 1977;23:19–22.

Tassone EE, Geib SM, Hall B, Fabrick JA, Brent CS, Hull JJ. De novo construction of an expanded transcriptome assembly for the western tarnished plant bug, Lygus hesperus. Gigascience. 2016;5:6. PubMed PMC

Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10–2. doi: 10.14806/ej.17.1.200. DOI

Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29:644–52. doi: 10.1038/nbt.1883. PubMed DOI PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–20. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. doi: 10.1038/nmeth.1923. PubMed DOI PMC

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, 1000 Genome Project Data Processing Subgroup The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009;25:2078–9. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Milne I, Stephen G, Bayer M, Cock PJ, Pritchard L, Cardle L, Shaw PD, Marshall D. Using tablet for visual exploration of second-generation sequencing data. Brief Bioinform. 2013;14:193–202. doi: 10.1093/bib/bbs012. PubMed DOI

Thorvaldsdóttir H, Robinson JT, Mesirov JP. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013;14:178–92. doi: 10.1093/bib/bbs017. PubMed DOI PMC

Burki F, Kaplan M, Tikhonenkov DV, Zlatogursky V, Minh BQ, Radaykina LV, Smirnov A, Mylnikov AP, Keeling PJ. Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida. Haptophyta and Cryptista. Proc Biol Sci. 2016;283:20152802. doi: 10.1098/rspb.2015.2802. PubMed DOI PMC

Sierra R, Cañas-Duarte SJ, Burki F, Schwelm A, Fogelqvist J, Dixelius C, González-García LN, Gile GH, Slamovits CH, Klopp C, et al. Evolutionary origins of rhizarian parasites. Mol Biol Evol. 2016;33:980–3. doi: 10.1093/molbev/msv340. PubMed DOI

Pánek T, Simpson AGB, Hampl V, Cepicka I. Creneis carolina gen. et sp. nov. (Heterolobosea), a novel marine anaerobic protist with strikingly derived morphology and life cycle. Protist. 2014;165:542–67. PubMed

Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013;30:772–80. doi: 10.1093/molbev/mst010. PubMed DOI PMC

Vaidya G, Lohman DJ, Meier R. SequenceMatrix: concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics. 2011;27:171–80. doi: 10.1111/j.1096-0031.2010.00329.x. PubMed DOI

Stamatakis A. RAxML Version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014;30:1312–3. doi: 10.1093/bioinformatics/btu033. PubMed DOI PMC

Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE). New Orleans; 2010. p. 1–8.

Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015;32:268–74. doi: 10.1093/molbev/msu300. PubMed DOI PMC

Wang HC, Susko E, Roger AJ. An amino acid substitution-selection model adjusts residue fitness to improve phylogenetic estimation. Mol Biol Evol. 2014;31:779–92. doi: 10.1093/molbev/msu044. PubMed DOI

Lartillot N, Rodrigue N, Stubbs D, Richer J. PhyloBayes MPI: phylogenetic reconstruction with infinite mixtures of profiles in a parallel environment. Syst Biol. 2013;62:611–5. doi: 10.1093/sysbio/syt022. PubMed DOI

RStudio Team. RStudio: Integrated Development for R. Boston: RStudio Inc.; 2015. http://www.rstudio.com. Accessed 21 Aug 2016.

Derelle R, Torruella G, Klimeš V, Brinkmann H, Kim E, Vlček Č, Lang BF, Eliáš M. Bacterial proteins pinpoint a single eukaryotic root. Proc Natl Acad Sci U S A. 2015;112:E693–9. doi: 10.1073/pnas.1420657112. PubMed DOI PMC

Karnkowska A, Vacek V, Zubáčová Z, Treitli SC, Petrželková R, Eme L, Novák L, Žárský V, Barlow LD, Herman EK, et al. A eukaryote without a mitochondrial organelle. Curr Biol. 2016;26:1274–84. doi: 10.1016/j.cub.2016.03.053. PubMed DOI

Gentekaki E, Kolisko M, Boscaro V, Bright KJ, Dini F, Di Giuseppe G, Gong Y, Miceli C, Modeo L, Molestina RE, et al. Large-scale phylogenomic analysis reveals the phylogenetic position of the problematic taxon Protocruzia and unravels the deep phylogenetic affinities of the ciliate lineages. Mol Phylogenet Evol. 2014;78:36–42. PubMed

Chen X, Zhao X, Liu X, Warren A, Zhao F, Miao M. Phylogenomics of non-model ciliates based on transcriptomic analyses. Protein Cell. 2015;6:373–85. doi: 10.1007/s13238-015-0147-3. PubMed DOI PMC

Feng JM, Jiang CQ, Warren A, Tian M, Cheng J, Liu GL, Xiong J, Miao W. Phylogenomic analyses reveal subclass Scuticociliatia as the sister group of subclass Hymenostomatia within class Oligohymenophorea. Mol Phylogenet Evol. 2015;90:104–11. doi: 10.1016/j.ympev.2015.05.007. PubMed DOI

Fernandes NM, Paiva Tda S, da Silva-Neto ID, Schlegel M, Schrago CG. Expanded phylogenetic analyses of the class Heterotrichea (Ciliophora, Postciliodesmatophora) using five molecular markers and morphological data. Mol Phylogenet Evol. 2016;95:229–46. doi: 10.1016/j.ympev.2015.10.030. PubMed DOI

Gao F, Warren A, Zhang Q, Gong J, Miao M, Sun P, Xu D, Huang J, Yi Z, Song W. The all-data-based evolutionary hypothesis of ciliated protists with a revised classification of the phylum Ciliophora (Eukaryota, Alveolata) Sci Rep. 2016;6:24874. doi: 10.1038/srep24874. PubMed DOI PMC

Ribosomal A-site interactions with near-cognate tRNAs drive stop codon readthrough

Evidence for an Independent Hydrogenosome-to-Mitosome Transition in the CL3 Lineage of Fornicates

Evolution and Unprecedented Variants of the Mitochondrial Genetic Code in a Lineage of Green Algae