Noncoding introns are a unifying feature of protein-coding genes in virtually all extant eukaryotes, with most lineages following the canonical intron structure. However, euglenozoans, unicellular flagellates that include free-living euglenids, human pathogenic kinetoplastids, and highly diverse and abundant marine diplonemids, are a notable exception. Euglenozoan genomes range from extremely intron-poor kinetoplastids to euglenid genomes containing both canonical and noncanonical introns. Here, we present a comprehensive analysis of splice sites and spliceosomal components in six species of understudied diplonemids. All diplonemids examined contain a nearly complete set of spliceosomal snRNP components, indicating the presence of a functional U2-type spliceosome. However, the majority of introns in the hemistasiid diplonemids Artemidia motanka and Namystynia karyoxenos are noncanonical and lack conserved GT-AG terminal dinucleotides typical for U2-type introns. These noncanonical introns are capable of extensive base-pairing, which brings intron ends into close proximity. Thus, while the splicing apparatus is conserved in diplonemids, the splice sites are highly variable among individual species.

Faculty of Science University of South Bohemia České Budějovice 370 05 Czech Republic

Institute of Molecular Genetics Czech Academy of Sciences Prague 142 20 Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences České Budějovice 370 05 Czech Republic

Life Science Research Centre Faculty of Science University of Ostrava Ostrava 701 03 Czech Republic

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Akinyi MV, Frilander MJ. 2021. At the intersection of major and minor spliceosomes: crosstalk mechanisms and their impact on gene expression. Front Genet 12: 700744. 10.3389/fgene.2021.700744 PubMed DOI PMC

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215: 403–410. 10.1016/S0022-2836(05)80360-2 PubMed DOI

Bailey TL, Elkan C. 1994. Fitting a mixture model by expectation maximization to discover motifs in biopolymers. Proc Int Conf Intell Syst Mol Biol 2: 28–36. PubMed

Benz C, Raas MWD, Tripathi P, Faktorova D, Tromer EC, Akiyoshi B, Lukes J. 2024. On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa. mBio 15: e0293624. 10.1128/mbio.02936-24 PubMed DOI PMC

Breckenridge DG, Watanabe Y, Greenwood SJ, Gray MW, Schnare MN. 1999. U1 small nuclear RNA and spliceosomal introns in PubMed DOI PMC

Brun R, Schonenberger. 1979. Cultivation and in vitro cloning or procyclic culture forms of PubMed

Burger G, Valach M. 2018. Perfection of eccentricity: mitochondrial genomes of diplonemids. IUBMB Life 70: 1197–1206. 10.1002/iub.1927 PubMed DOI

Bushmanova E, Antipov D, Lapidus A, Prjibelski AD. 2019. rnaSPAdes: a PubMed DOI PMC

Bushnell B, Rood J, Singer E. 2017. BBMerge - accurate paired shotgun read merging via overlap. PLoS One 12: e0185056. 10.1371/journal.pone.0185056 PubMed DOI PMC

Crooks GE, Hon G, Chandonia JM, Brenner SE. 2004. WebLogo: a sequence logo generator. Genome Res 14: 1188–1190. 10.1101/gr.849004 PubMed DOI PMC

Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR. 2013. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29: 15–21. 10.1093/bioinformatics/bts635 PubMed DOI PMC

Emms DM, Kelly S. 2017. STRIDE: species tree root inference from gene duplication events. Mol Biol Evol 34: 3267–3278. 10.1093/molbev/msx259 PubMed DOI PMC

Emms DM, Kelly S. 2018. STAG: species tree inference from all genes. bioRxiv 10.1101/267914 DOI

Emms DM, Kelly S. 2019. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 20: 238. 10.1186/s13059-019-1832-y PubMed DOI PMC

Faktorová D, Kaur B, Valach M, Graf L, Benz C, Burger G, Lukes J. 2020. Targeted integration by homologous recombination enables PubMed DOI

Faktorová D, Zahonova K, Benz C, Dacks JB, Field MC, Lukes J. 2023. Functional differentiation of Sec13 paralogues in the euglenozoan protists. Open Biol 13: 220364. 10.1098/rsob.220364 PubMed DOI PMC

Feschotte C, Pritham EJ. 2007. DNA transposons and the evolution of eukaryotic genomes. Annu Rev Genet 41: 331–368. 10.1146/annurev.genet.40.110405.090448 PubMed DOI PMC

Flegontova O, Flegontov P, Malviya S, Audic S, Wincker P, de Vargas C, Bowler C, Lukes J, Horak A. 2016. Extreme diversity of diplonemid eukaryotes in the ocean. Curr Biol 26: 3060–3065. 10.1016/j.cub.2016.09.031 PubMed DOI

Flegontova O, Flegontov P, Londono PAC, Walczowski W, Santic D, Edgcomb VP, Lukes J, Horak A. 2020. Environmental determinants of the distribution of planktonic diplonemids and kinetoplastids in the oceans. Environ Microbiol 22: 4014–4031. 10.1111/1462-2920.15190 PubMed DOI

Gawryluk RMR, Del Campo J, Okamoto N, Strassert JFH, Lukes J, Richards TA, Worden AZ, Santoro AE, Keeling PJ. 2016. Morphological identification and single-cell genomics of marine diplonemids. Curr Biol 26: 3053–3059. 10.1016/j.cub.2016.09.013 PubMed DOI

Gerber JL, Köhler S, Peschek J. 2022. Eukaryotic tRNA splicing - one goal, two strategies, many players. Biol Chem 403: 765–778. 10.1515/hsz-2021-0402 PubMed DOI

Guminska N, Zakrys B, Milanowski R. 2021. A new type of circular RNA derived from nonconventional introns in nuclear genes of euglenids. J Mol Biol 433: 166758. 10.1016/j.jmb.2020.166758 PubMed DOI

Henriet S, Colom Sanmarti B, Sumic S, Chourrout D. 2019. Evolution of the U2 spliceosome for processing numerous and highly diverse non-canonical introns in the chordate PubMed DOI

Henze K, Badr A, Wettern M, Cerff R, Martin W. 1995. A nuclear gene of eubacterial origin in PubMed DOI PMC

Kastner B, Will CL, Stark H, Lührmann R. 2019. Structural insights into nuclear pre-mRNA splicing in higher eukaryotes. CSH Perspect Biol 11: a032417. 10.1101/cshperspect.a032417 PubMed DOI PMC

Kaur B, Valach M, Pena-Diaz P, Moreira S, Keeling PJ, Burger G, Lukes J, Faktorova D. 2018. Transformation of PubMed DOI

Kaur B, Zahonova K, Valach M, Faktorova D, Prokopchuk G, Burger G, Lukes J. 2020. Gene fragmentation and RNA editing without borders: eccentric mitochondrial genomes of diplonemids. Nucleic Acids Res 48: 2694–2708. 10.1093/nar/gkz1215 PubMed DOI PMC

Kazazian HH Jr. 2004. Mobile elements: drivers of genome evolution. Science 303: 1626–1632. 10.1126/science.1089670 PubMed DOI

Kostygov AY, Karnkowska A, Votypka J, Tashyreva D, Maciszewski K, Yurchenko V, Lukes J. 2021. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol 11: 200407. 10.1098/rsob.200407 PubMed DOI PMC

Kostygov AY, Skypalova K, Kraeva N, Kalita E, McLeod C, Yurchenko V, Field MC, Lukes J, Butenko A. 2024. Comprehensive analysis of the Kinetoplastea intron landscape reveals a novel intron-containing gene and the first exclusively PubMed DOI PMC

Krchnáková Z, Thakur PK, Krausová M, Bieberstein N, Haberman N, Müller-McNicoll M, Stanek D. 2019. Splicing of long non-coding RNAs primarily depends on polypyrimidine tract and 5 splice-site sequences due to weak interactions with SR proteins. Nucleic Acids Res 47: 911–928. 10.1093/nar/gky1147 PubMed DOI PMC

Letunic I, Bork P. 2021. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation. Nucleic Acids Res 49: W293–W296. 10.1093/nar/gkab301 PubMed DOI PMC

Lorenz R, Bernhart SH, Siederdissen CHZ, Tafer H, Flamm C, Stadler PF, Hofacker IL. 2011. ViennaRNA Package 2.0. Algorithm Mol Biol 6: 26. 10.1186/1748-7188-6-26 PubMed DOI PMC

Lu ZP, Filonov GS, Noto JJ, Schmidt CA, Hatkevich TL, Wen Y, Jaffrey SR, Matera AG. 2015. Metazoan tRNA introns generate stable circular RNAs in vivo. RNA 21: 1554–1565. 10.1261/rna.052944.115 PubMed DOI PMC

Lukeš J, Wheeler R, Jirsova D, David V, Archibald JM. 2018. Massive mitochondrial DNA content in diplonemid and kinetoplastid protists. IUBMB Life 70: 1267–1274. 10.1002/iub.1894 PubMed DOI PMC

Mair G, Shi H, Li H, Djikeng A, Aviles HO, Bishop JR, Falcone FH, Gavrilescu C, Montgomery JL, Santori MI, et al. 2000. A new twist in trypanosome RNA metabolism: PubMed DOI PMC

Mapleson D, Garcia Accinelli G, Kettleborough G, Wright J, Clavijo BJ. 2017. KAT: a K-mer analysis toolkit to quality control NGS datasets and genome assemblies. Bioinformatics 33: 574–576. 10.1093/bioinformatics/btw663 PubMed DOI PMC

McWatters DC, Russell AG. 2017. Euglena transcript processing. Adv Exp Med Biol 979: 141–158. 10.1007/978-3-319-54910-1_8 PubMed DOI

Milanowski R, Karnkowska A, Ishikawa T, Zakrys B. 2014. Distribution of conventional and nonconventional introns in tubulin (α and β) genes of euglenids. Mol Biol Evol 31: 584–593. 10.1093/molbev/mst227 PubMed DOI PMC

Milanowski R, Guminska N, Karnkowska A, Ishikawa T, Zakrys B. 2016. Intermediate introns in nuclear genes of euglenids - Are they a distinct type? BMC Evol Biol 16: 49. 10.1186/s12862-016-0620-5 PubMed DOI PMC

Min XJ, Butler G, Storms R, Tsang A. 2005. OrfPredictor: predicting protein-coding regions in EST-derived sequences. Nucleic Acids Res 33: W677–W680. 10.1093/nar/gki394 PubMed DOI PMC

Muchhal US, Schwartzbach SD. 1992. Characterization of a Euglena gene encoding a polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II. Plant Mol Biol 18: 287–299. 10.1007/BF00034956 PubMed DOI

Muchhal US, Schwartzbach SD. 1994. Characterization of the unique intron-exon junctions of Euglena gene(s) encoding the polyprotein precursor to the light-harvesting chlorophyll a/b binding protein of photosystem II. Nucleic Acids Res 22: 5737–5744. 10.1093/nar/22.25.5737 PubMed DOI PMC

Nawrocki EP, Eddy SR. 2013. Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics 29: 2933–2935. 10.1093/bioinformatics/btt509 PubMed DOI PMC

Nielsen H, Johansen SD. 2009. Group I introns: moving in new directions. RNA Biol 6: 375–383. 10.4161/rna.6.4.9334 PubMed DOI

Obiol A, Giner CR, Sanchez P, Duarte CM, Acinas SG, Massana R. 2020. A metagenomic assessment of microbial eukaryotic diversity in the global ocean. Mol Ecol Resour 20: 718–731. 10.1111/1755-0998.13147 PubMed DOI

Pagès H, Aboyoun P, Gentleman R, DebRoy S. 2024. Biostrings: efficient manipulation of biological strings. R package version 2.70.3. https://bioconductor.org/packages/Biostrings.

Paradis E, Schliep K. 2019. ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics 35: 526–528. 10.1093/bioinformatics/bty633 PubMed DOI

Patel AA, Steitz JA. 2003. Splicing double: insights from the second spliceosome. Nat Rev Mol Cell Biol 4: 960–970. 10.1038/nrm1259 PubMed DOI

Poon SK, Peacock L, Gibson W, Gull K, Kelly S. 2012. A modular and optimized single marker system for generating PubMed DOI PMC

Prjibelski A, Antipov D, Meleshko D, Lapidus A, Korobeynikov A. 2020. Using SPAdes PubMed DOI

Prokopchuk G, Tashyreva D, Yabuki A, Horak A, Masarova P, Lukes J. 2019. Morphological, ultrastructural, motility and evolutionary characterization of two new hemistasiidae species. Protist 170: 259–282. 10.1016/j.protis.2019.04.001 PubMed DOI

Pyle AM. 2016. Group II intron self-splicing. Annu Rev Biophys 45: 183–205. 10.1146/annurev-biophys-062215-011149 PubMed DOI

Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26: 841–842. 10.1093/bioinformatics/btq033 PubMed DOI PMC

R Core Team. 2021. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org.

Reuter JS, Mathews DH. 2010. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11: 129. 10.1186/1471-2105-11-129 PubMed DOI PMC

Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP. 2011. Integrative genomics viewer. Nat Biotechnol 29: 24–26. 10.1038/nbt.1754 PubMed DOI PMC

Schneider A, Martin J, Agabian N. 1994. A nuclear encoded tRNA of PubMed DOI PMC

Schoenle A, Hohlfeld M, Hermanns K, Mahé F, de Vargas C, Nitsche F, Arndt H. 2021. High and specific diversity of protists in the deep-sea basins dominated by diplonemids, kinetoplastids, ciliates and foraminiferans. Commun Biol 4: 501. 10.1038/s42003-021-02012-5 PubMed DOI PMC

Seppey M, Manni M, Zdobnov EM. 2019. BUSCO: assessing genome assembly and annotation completeness. Methods Mol Biol 1962: 227–245. 10.1007/978-1-4939-9173-0_14 PubMed DOI

Shen W, Le S, Li Y, Hu F. 2016. SeqKit: a cross-platform and ultrafast toolkit for FASTA/Q file manipulation. PLoS One 11: e0163962. 10.1371/journal.pone.0163962 PubMed DOI PMC

Tashyreva D, Prokopchuk G, Yabuki A, Kaur B, Faktorova D, Votypka J, Kusaka C, Fujikura K, Shiratori T, Ishida KI, et al. 2018. Phylogeny and morphology of new diplonemids from Japan. Protist 169: 158–179. 10.1016/j.protis.2018.02.001 PubMed DOI

Tashyreva D, Simpson AGB, Prokopchuk G, Skodová-Sveráková I, Butenko A, Hammond M, George EE, Flegontova O, Záhonová K, Faktorová D, et al. 2022. Diplonemids - a review on “new” flagellates on the oceanic block. Protist 173: 125868. 10.1016/j.protis.2022.125868 PubMed DOI

Tessier LH, Paulus F, Keller M, Vial C, Imbault P. 1995. Structure and expression of PubMed DOI

Thompson JD, Gibson TJ, Higgins DG. 2002. Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2: Unit 2.3. 10.1002/0471250953.bi0203s00 PubMed DOI

Valach M, Benz C, Aguilar LC, Gahura O, Faktorová D, Zíková A, Oeffinger M, Burger G, Gray MW, Lukes J. 2023a. Miniature RNAs are embedded in an exceptionally protein-rich mitoribosome via an elaborate assembly pathway. Nucleic Acids Res 51: 6443–6460. 10.1093/nar/gkad422 PubMed DOI PMC

Valach M, Moreira S, Petitjean C, Benz C, Butenko A, Flegontova O, Nenarokova A, Prokopchuk G, Batstone T, Lapebie P, et al. 2023b. Recent expansion of metabolic versatility in PubMed DOI PMC

Waterhouse RM, Seppey M, Simao FA, Zdobnov EM. 2019. Using BUSCO to assess insect genomic resources. Methods Mol Biol 1858: 59–74. 10.1007/978-1-4939-8775-7_6 PubMed DOI

Weinberg Z, Breaker RR. 2011. R2R - software to speed the depiction of aesthetic consensus RNA secondary structures. BMC Bioinformatics 12: 3. 10.1186/1471-2105-12-3 PubMed DOI PMC

Wilkinson ME, Charenton C, Nagai K. 2020. RNA splicing by the spliceosome. Annu Rev Biochem 89: 359–388. 10.1146/annurev-biochem-091719-064225 PubMed DOI

Will CL, Lührmann R. 2011. Spliceosome structure and function. CSH Perspect Biol 3: a003707. 10.1101/cshperspect.a003707 PubMed DOI PMC

Wirtz E, Leal S, Ochatt C, Cross GA. 1999. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in PubMed DOI

Wu TD, Watanabe CK. 2005. GMAP: a genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21: 1859–1875. 10.1093/bioinformatics/bti310 PubMed DOI

Yeo G, Burge CB. 2004. Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J Comput Biol 11: 377–394. 10.1089/1066527041410418 PubMed DOI

Záhonová K, Lukeš J, Dacks JB. 2025. Diplonemid protists possess exotic endomembrane machinery, impacting models of membrane trafficking in modern and ancient eukaryotes. Curr Biol 35: 1508–1520.e2. 10.1016/j.cub.2025.02.032 PubMed DOI