BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

• Klíčová slova
• CAZymes, Ecological distribution, Feeding strategy, Gene-family evolution, Genome, Geographical distribution, Lateral gene transfer, Paradiplonema papillatum, Proteome, Protists, Transcriptome,
• MeSH
• Euglenozoa genetika   MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• Kinetoplastida * genetika   MeSH
• lidé MeSH
• multigenová rodina MeSH
• profáze meiózy I MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Diplonemids are highly abundant heterotrophic marine protists. Previous studies showed that their strikingly bloated mitochondrial genome is unique because of systematic gene fragmentation and manifold RNA editing. Here we report a comparative study of mitochondrial genome architecture, gene structure and RNA editing of six recently isolated, phylogenetically diverse diplonemid species. Mitochondrial gene fragmentation and modes of RNA editing, which include cytidine-to-uridine (C-to-U) and adenosine-to-inosine (A-to-I) substitutions and 3' uridine additions (U-appendage), are conserved across diplonemids. Yet as we show here, all these features have been pushed to their extremes in the Hemistasiidae lineage. For example, Namystynia karyoxenos has its genes fragmented into more than twice as many modules than other diplonemids, with modules as short as four nucleotides. Furthermore, we detected in this group multiple A-appendage and guanosine-to-adenosine (G-to-A) substitution editing events not observed before in diplonemids and found very rarely elsewhere. With >1,000 sites, C-to-U and A-to-I editing in Namystynia is nearly 10 times more frequent than in other diplonemids. The editing density of 12% in coding regions makes Namystynia's the most extensively edited transcriptome described so far. Diplonemid mitochondrial genome architecture, gene structure and post-transcriptional processes display such high complexity that they challenge all other currently known systems.

• MeSH
• chromozomy genetika   MeSH
• editace RNA genetika   MeSH
• Euglenozoa genetika   MeSH
• fylogeneze MeSH
• genom mitochondriální * MeSH
• geny * MeSH
• konzervovaná sekvence MeSH
• mitochondriální DNA genetika   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

The instructions to make proteins and structural RNAs are laid down in gene sequences. Yet, in certain instances, these primary instructions need to be modified considerably during gene expression, most often at the transcript level. Here we review a case of massive post-transcriptional revisions via trans-splicing and RNA editing, a phenomenon occurring in mitochondria of a recently recognized protist group, the diplonemids. As of now, the various post-transcriptional steps have been cataloged in detail, but how these processes function is still unknown. Since genetic manipulation techniques such as gene replacement and RNA interference have not yet been established for these organisms, alternative strategies have to be deployed. Here, we discuss the experimental and bioinformatics approaches that promise to unravel the molecular machineries of trans-splicing and RNA editing in Diplonema mitochondria.

• Klíčová slova
• Cryptic genes, RNA editing, diplonemids, gene fragmentation, multipartite mtDNA, trans-splicing,
• MeSH
• editace RNA MeSH
• Euglenozoa genetika   MeSH
• mitochondriální proteiny genetika   MeSH
• mitochondrie genetika   MeSH
• posttranskripční úpravy RNA MeSH
• protozoální proteiny genetika   MeSH
• regulace genové exprese MeSH
• RNA transferová metabolismus   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• protozoální proteiny MeSH
• RNA transferová MeSH

Mitochondria are double membrane organelles of endosymbiotic origin, best known for constituting the centre of energetics of a eukaryotic cell. They contain their own mitochondrial genome, which as a consequence of gradual reduction during evolution typically contains less than two dozens of genes. In this review, we highlight the extremely diverse architecture of mitochondrial genomes and mechanisms of gene expression between the three sister groups constituting the phylum Euglenozoa - Euglenida, Diplonemea and Kinetoplastea. The earliest diverging euglenids possess a simplified mitochondrial genome and a conventional gene expression, whereas both are highly complex in the two other groups. The expression of their mitochondrial-encoded proteins requires extensive post-transcriptional modifications guided by complex protein machineries and multiple small RNA molecules. Moreover, the least studied diplonemids, which have been recently discovered as a highly abundant component of the world ocean plankton, possess one of the most complicated mitochondrial genome organisations known to date.

• Klíčová slova
• euglenozoa, mitochondria, mitochondrial genome,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

UNLABELLED: Perkinsela is an enigmatic early-branching kinetoplastid protist that lives as an obligate endosymbiont inside Paramoeba (Amoebozoa). We have sequenced the highly reduced mitochondrial genome of Perkinsela, which possesses only six protein-coding genes (cox1, cox2, cox3, cob, atp6, and rps12), despite the fact that the organelle itself contains more DNA than is present in either the host or endosymbiont nuclear genomes. An in silico analysis of two Perkinsela strains showed that mitochondrial RNA editing and processing machineries typical of kinetoplastid flagellates are generally conserved, and all mitochondrial transcripts undergo U-insertion/deletion editing. Canonical kinetoplastid mitochondrial ribosomes are also present. We have developed software tools for accurate and exhaustive mapping of transcriptome sequencing (RNA-seq) reads with extensive U-insertions/deletions, which allows detailed investigation of RNA editing via deep sequencing. With these methods, we show that up to 50% of reads for a given edited region contain errors of the editing system or, less likely, correspond to alternatively edited transcripts. IMPORTANCE: Uridine insertion/deletion-type RNA editing, which occurs in the mitochondrion of kinetoplastid protists, has been well-studied in the model parasite genera Trypanosoma, Leishmania, and Crithidia. Perkinsela provides a unique opportunity to broaden our knowledge of RNA editing machinery from an evolutionary perspective, as it represents the earliest kinetoplastid branch and is an obligatory endosymbiont with extensive reductive trends. Interestingly, up to 50% of mitochondrial transcripts in Perkinsela contain errors. Our study was complemented by use of newly developed software designed for accurate mapping of extensively edited RNA-seq reads obtained by deep sequencing.

• MeSH
• Amoebozoa parazitologie   MeSH
• delece genu * MeSH
• editace RNA * MeSH
• Kinetoplastida genetika   růst a vývoj   MeSH
• mitochondriální DNA chemie   genetika   MeSH
• mitochondrie genetika   MeSH
• sekvenční analýza DNA MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální DNA MeSH

In this study, we describe the mitochondrial genome of the excavate flagellate Euglena gracilis. Its gene complement is reduced as compared with the well-studied sister groups Diplonemea and Kinetoplastea. We have identified seven protein-coding genes: Three subunits of respiratory complex I (nad1, nad4, and nad5), one subunit of complex III (cob), and three subunits of complex IV (cox1, cox2, and a highly divergent cox3). Moreover, fragments of ribosomal RNA genes have also been identified. Genes encoding subunits of complex V, ribosomal proteins and tRNAs were missing, and are likely located in the nuclear genome. Although mitochondrial genomes of diplonemids and kinetoplastids possess the most complex RNA processing machineries known, including trans-splicing and editing of the uridine insertion/deletion type, respectively, our transcriptomic data suggest their total absence in E. gracilis. This finding supports a scenario in which the complex mitochondrial processing machineries of both sister groups evolved relatively late in evolution from a streamlined genome and transcriptome of their common predecessor.

• Klíčová slova
• Euglena gracilis, RNA editing, mitochondrial genome, transcription,
• MeSH
• editace RNA MeSH
• elektronový transportní řetězec genetika   MeSH
• Euglena gracilis genetika   MeSH
• genom mitochondriální * MeSH
• molekulární evoluce * MeSH
• RNA ribozomální genetika   MeSH
• sestřih RNA MeSH
• transkriptom MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• elektronový transportní řetězec MeSH
• RNA ribozomální MeSH

Termination codons in mRNA molecules are typically specified directly by the sequence of the corresponding gene. However, in mitochondria of a few eukaryotic groups, some mRNAs contain the termination codon UAA deriving one or both adenosines from transcript polyadenylation. Here, we show that a similar phenomenon occurs for a substantial number of nuclear genes in Blastocystis spp., divergent unicellular eukaryote gut parasites. Our analyses of published genomic data from Blastocystis sp. subtype 7 revealed that polyadenylation-mediated creation of termination codons occurs in approximately 15% of all nuclear genes. As this phenomenon has not been noticed before, the procedure previously employed to annotate the Blastocystis nuclear genome sequence failed to correctly define the structure of the 3'-ends of hundreds of genes. From sequence data we have obtained from the distantly related Blastocystis sp. subtype 1 strain, we show that this phenomenon is widespread within the Blastocystis genus. Polyadenylation in Blastocystis appears to be directed by a conserved GU-rich element located four nucleotides downstream of the polyadenylation site. Thus, the highly precise positioning of the polyadenylation in Blastocystis has allowed reduction of the 3'-untranslated regions to the point that, in many genes, only one or two nucleotides of the termination codon are left.

• Klíčová slova
• Blastocystis, evolution, gene expression, mRNA processing, polyadenylation, termination codons, translation,
• MeSH
• Blastocystis chemie   genetika   MeSH
• blastocystóza parazitologie   MeSH
• lidé MeSH
• messenger RNA chemie   genetika   MeSH
• molekulární sekvence - údaje MeSH
• polyadenylace * MeSH
• protozoální proteiny chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• terminační kodon chemie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• protozoální proteiny MeSH
• terminační kodon MeSH