UNLABELLED: Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates-kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved among most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid or canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, Paradiplonema papillatum. PpCENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. PpMad2, PpCLKKKT10/19, PpSYCP2L1KKT17/18, and PpINCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system. IMPORTANCE: A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplonemids except for a putative centromere-specific histone H3 variant. Our results suggest that diplonemids evolved kinetochores that do not resemble previously known ones.
The mitochondrial ribosome (mitoribosome) has diverged drastically from its evolutionary progenitor, the bacterial ribosome. Structural and compositional diversity is particularly striking in the phylum Euglenozoa, with an extraordinary protein gain in the mitoribosome of kinetoplastid protists. Here we report an even more complex mitoribosome in diplonemids, the sister-group of kinetoplastids. Affinity pulldown of mitoribosomal complexes from Diplonema papillatum, the diplonemid type species, demonstrates that they have a mass of > 5 MDa, contain as many as 130 integral proteins, and exhibit a protein-to-RNA ratio of 11:1. This unusual composition reflects unprecedented structural reduction of ribosomal RNAs, increased size of canonical mitoribosomal proteins, and accretion of three dozen lineage-specific components. In addition, we identified >50 candidate assembly factors, around half of which contribute to early mitoribosome maturation steps. Because little is known about early assembly stages even in model organisms, our investigation of the diplonemid mitoribosome illuminates this process. Together, our results provide a foundation for understanding how runaway evolutionary divergence shapes both biogenesis and function of a complex molecular machine.
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.
- 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
The unicellular trypanosomatids belong to the phylum Euglenozoa and all known species are obligate parasites. Distinct lineages infect plants, invertebrates, and vertebrates, including humans. Genome data for marine diplonemids, together with freshwater euglenids and free-living kinetoplastids, the closest known nonparasitic relatives to trypanosomatids, recently became available. Robust phylogenetic reconstructions across Euglenozoa are now possible and place the results of parasite-focused studies into an evolutionary context. Here we discuss recent advances in identifying the factors shaping the evolution of Euglenozoa, focusing on ancestral features generally considered parasite-specific. Remarkably, most of these predate the transition(s) to parasitism, suggesting that the presence of certain preconditions makes a significant lifestyle change more likely.
- MeSH
- biologická evoluce * MeSH
- datové soubory jako téma MeSH
- Euglenozoa klasifikace genetika MeSH
- fylogeneze MeSH
- genom genetika MeSH
- infekce prvoky kmene Euglenozoa parazitologie MeSH
- lidé MeSH
- paraziti klasifikace genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy 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.
BACKGROUND: The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. RESULTS: We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. CONCLUSIONS: The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.
Recent surveys of marine microbial diversity have identified a previously unrecognized lineage of diplonemid protists as being among the most diverse heterotrophic eukaryotes in global oceans. Despite their monophyly (and assumed importance), they lack a formal taxonomic description, and are informally known as deep-sea pelagic diplonemids (DSPDs) or marine diplonemids. Recently, we documented morphology and molecular sequences from several DSPDs, one of which is particularly widespread and abundant in environmental sequence data. To simplify the communication of future work on this important group, here we formally propose to erect the family Eupelagonemidae to encompass this clade, as well as a formal genus and species description for the apparently most abundant phylotype, Eupelagonema oceanica, for which morphological information and single-cell amplified genome data are currently available.
- MeSH
- Euglenozoa klasifikace cytologie genetika MeSH
- fylogeneze MeSH
- RNA protozoální analýza MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (β-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.
- MeSH
- biologická evoluce * MeSH
- DNA řízené RNA-polymerasy genetika metabolismus MeSH
- Euglenida klasifikace genetika MeSH
- Euglenozoa klasifikace genetika MeSH
- fototrofní procesy MeSH
- fylogeneze MeSH
- genom fyziologie MeSH
- introny fyziologie MeSH
- mitochondrie genetika MeSH
- molekulární biologie * MeSH
- RNA interference MeSH
- RNA ribozomální 28S genetika MeSH
- Trypanosomatina klasifikace enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
The mitochondrial DNA of diplonemid and kinetoplastid protists is known for its suite of bizarre features, including the presence of concatenated circular molecules, extensive trans-splicing and various forms of RNA editing. Here we report on the existence of another remarkable characteristic: hyper-inflated DNA content. We estimated the total amount of mitochondrial DNA in four kinetoplastid species (Trypanosoma brucei, Trypanoplasma borreli, Cryptobia helicis, and Perkinsela sp.) and the diplonemid Diplonema papillatum. Staining with 4',6-diamidino-2-phenylindole and RedDot1 followed by color deconvolution and quantification revealed massive inflation in the total amount of DNA in their organelles. This was further confirmed by electron microscopy. The most extreme case is the ∼260 Mbp of DNA in the mitochondrion of Diplonema, which greatly exceeds that in its nucleus; this is, to our knowledge, the largest amount of DNA described in any organelle. Perkinsela sp. has a total mitochondrial DNA content ~6.6× greater than its nuclear genome. This mass of DNA occupies most of the volume of the Perkinsela cell, despite the fact that it contains only six protein-coding genes. Why so much DNA? We propose that these bloated mitochondrial DNAs accumulated by a ratchet-like process. Despite their excessive nature, the synthesis and maintenance of these mtDNAs must incur a relatively low cost, considering that diplonemids are one of the most ubiquitous and speciose protist groups in the ocean. © 2018 IUBMB Life, 70(12):1267-1274, 2018.
Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering.
- MeSH
- buněčná membrána metabolismus MeSH
- Euglenozoa genetika metabolismus MeSH
- fylogeneze * MeSH
- infekce prvoky kmene Euglenozoa parazitologie MeSH
- karcinogeneze genetika metabolismus MeSH
- lidé MeSH
- molekulární evoluce MeSH
- protein Rheb chemie genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
