Diplonemids are highly diverse and abundant marine plankton with significant ecological importance. However, little is known about their biology, even in the model diplonemid Paradiplonema papillatum whose genome sequence is available. Examining the subcellular localization of proteins using fluorescence microscopy is a powerful approach to infer their putative function. Here, we report a plasmid-based method that enables YFP-tagging of a gene at the endogenous locus. By examining the localization of proteins whose homologs are involved in chromosome organization or segregation in other eukaryotes, we discovered several notable features in mitotically dividing P. papillatum cells. Cohesin is enriched on condensed interphase chromatin. During mitosis, chromosomes organize into two rings (termed mitotic rings herein) that surround the elongating nucleolus and align on a bipolar spindle. Homologs of chromosomal passenger complex components (INCENP, two Aurora kinases and KIN-A), a CLK1 kinase, meiotic chromosome axis protein SYCP2L1, spindle checkpoint protein Mad1 and microtubule regulator XMAP215 localize in between the two mitotic rings. In contrast, a Mad2 homolog localizes near basal bodies as in trypanosomes. By representing the first molecular characterization of mitotic mechanisms in P. papillatum and raising many questions, this study forms the foundation for dissecting mitotic mechanisms in diplonemids.

• Klíčová slova
• Euglenozoa, chromosome, diplonemid, kinetochore, kinetoplastid,
• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• chromozomy metabolismus   MeSH
• Dinoflagellata * genetika   metabolismus   cytologie   MeSH
• mitóza * MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• segregace chromozomů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• proteiny buněčného cyklu MeSH

Arf and Rab family small GTPases and their regulators, GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs), play a central role in membrane trafficking. In this study, we focused on a recently reported GAP for Arf (and potentially Rab) proteins, the CSW complex, a part of a small family of longin domain-containing proteins that form complexes with GAP activity. This family also includes folliculin and GATOR1, which are GAPs for the Rag/Gtr GTPases. All three complexes are associated with lysosomes and play a role in nutrient signaling, the latter two being directly involved in the mTOR pathway. The role of CSW is not clear, but in addition to having GAP activity on Arf proteins in vitro, its mutation causes severe neurodegenerative diseases. Here we update the reported pan-eukaryotic presence of folliculin and GATOR1, and demonstrate that CSW is also found throughout eukaryotes, though with sporadic distribution. We identify highly conserved motifs in all CSW subunits, some shared with the catalytic subunits of folliculin and GATOR1, that provide new potential avenues for experimental exploration. Remarkably, one such conserved sequence, the "GP" motif, is also found in structurally related longin proteins present in the archaeal ancestor of eukaryotes.

• Klíčová slova
• Arf GTPases, DENN domain, GTPase‐activating proteins, LECA, molecular phylogenetics, nutrient signaling, pan‐eukaryotic homology search, structural modeling,
• MeSH
• lidé MeSH
• monomerní proteiny vázající GTP * metabolismus   MeSH
• proteinové domény MeSH
• proteiny aktivující GTPasu * metabolismus   genetika   chemie   MeSH
• výměnné faktory guaninnukleotidů metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• monomerní proteiny vázající GTP * MeSH
• proteiny aktivující GTPasu * MeSH
• výměnné faktory guaninnukleotidů MeSH

We present a genome assembly of the diplonemid Rhynchopus euleeides (Euglenozoa; Diplonemea; Diplonemea; Diplonemidae). The genome sequence is 199.0 megabases long, with most of the assembly scaffolded into 88 chromosomal pseudomolecules. The multipartite mitochondrial genome and the 2.0 megabase genome of Ca. Syngnamydia salmonis, a bacterial endosymbiont of R. euleeides, were also sequenced and assembled.

• Klíčová slova
• Diplonemea, Euglenozoa, Rhynchopus euleeides, bacterial endosymbionts, chromosomal, genome sequence,
• Publikační typ
• časopisecké články MeSH

Transposable elements (TEs) have the ability to move and amplify inside the host genome, making them a pivotal source of genome plasticity. Presently, only 4 TE clades (all classified as Class I retrotransposons) have been identified in trypanosomatids. We predicted repeat content and manually curated TEs across the genomes of 57 trypanosomatids, shedding light on their proportions, diversity and dynamics. Our analysis yielded 214 TE consensus sequence models across the dataset, with abundance ranging from 0.1% to 7.2%. We found evidence of recent transposon activity in most species, with notable bursts in the Vickermania, Lafontella, Porcisia and Angomonas spp., along with Leishmania (Mundinia) chancei, L. (M.) orientalis and L. (M.) procaviensis. We confirmed that the 4 TE clades have colonized virtually all lineages of trypanosomatids, potentially playing a role in shaping their genome architecture. The effort of this work culminated in the establishment of the Trypanosomatid TE Database 1.0, a resource designed to standardize the TE annotation process that can serve as a foundation for future studies on trypanosomatid TEs.

• Klíčová slova
• CRE, INGI, SLACS, TATE, VIPER, mobilome, transposable elements, trypanosomatids,
• MeSH
• fylogeneze MeSH
• genetická variace * MeSH
• genom protozoální * MeSH
• molekulární evoluce * MeSH
• retroelementy genetika   MeSH
• transpozibilní elementy DNA * genetika   MeSH
• Trypanosomatina * genetika   klasifikace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• retroelementy MeSH
• transpozibilní elementy DNA * MeSH

Connections between the mechanical properties of DNA and biological functions have been speculative due to the lack of methods to measure or predict DNA mechanics at scale. Recently, a proxy for DNA mechanics, cyclizability, was measured by loop-seq and enabled genome-scale investigation of DNA mechanics. Here, we use this dataset to build a computational model predicting bias-corrected intrinsic cyclizability, with near-perfect accuracy, solely based on DNA sequence. Further, the model predicts intrinsic bending direction in 3D space. Using this tool, we aimed to probe mechanical selection - that is, the evolutionary selection of DNA sequence based on its mechanical properties - in diverse circumstances. First, we found that the intrinsic bend direction of DNA sequences correlated with the observed bending in known protein-DNA complex structures, suggesting that many proteins co-evolved with their DNA partners to capture DNA in its intrinsically preferred bent conformation. We then applied our model to large-scale yeast population genetics data and showed that centromere DNA element II, whose consensus sequence is unknown, leaving its sequence-specific role unclear, is under mechanical selection to increase the stability of inner-kinetochore structure and to facilitate centromeric histone recruitment. Finally, in silico evolution under strong mechanical selection discovered hallucinated sequences with cyclizability values so extreme that they required experimental validation, yet, found in nature in the densely packed mitochondrial(mt) DNA of Namystynia karyoxenos, an ocean-dwelling protist with extreme mitochondrial gene fragmentation. The need to transmit an extraordinarily large amount of mtDNA, estimated to be > 600 Mb, in combination with the absence of mtDNA compaction proteins may have pushed mechanical selection to the extreme. Similarly extreme DNA mechanics are observed in bird microchromosomes, although the functional consequence is not yet clear. The discovery of eccentric DNA mechanics in unrelated unicellular and multicellular eukaryotes suggests that we can predict extreme natural biology which can arise through strong selection. Our methods offer a way to study the biological functions of DNA mechanics in any genome and to engineer DNA sequences with desired mechanical properties.

• Publikační typ
• časopisecké články MeSH
• preprinty MeSH

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.

• Klíčová slova
• Diplonemea, Kinetoplastea, Paradiplonema, cell division, cenH3/CENP-A, kinetochore,
• MeSH
• Euglenozoa * genetika   metabolismus   MeSH
• fylogeneze MeSH
• kinetochory * metabolismus   MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• segregace chromozomů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální proteiny MeSH

BACKGROUND: In trypanosomatids, a group of unicellular eukaryotes that includes numerous important human parasites, cis-splicing has been previously reported for only two genes: a poly(A) polymerase and an RNA helicase. Conversely, trans-splicing, which involves the attachment of a spliced leader sequence, is observed for nearly every protein-coding transcript. So far, our understanding of splicing in this protistan group has stemmed from the analysis of only a few medically relevant species. In this study, we used an extensive dataset encompassing all described trypanosomatid genera to investigate the distribution of intron-containing genes and the evolution of splice sites. RESULTS: We identified a new conserved intron-containing gene encoding an RNA-binding protein that is universally present in Kinetoplastea. We show that Perkinsela sp., a kinetoplastid endosymbiont of Amoebozoa, represents the first eukaryote completely devoid of cis-splicing, yet still preserving trans-splicing. We also provided evidence for reverse transcriptase-mediated intron loss in Kinetoplastea, extensive conservation of 5' splice sites, and the presence of non-coding RNAs within a subset of retained trypanosomatid introns. CONCLUSIONS: All three intron-containing genes identified in Kinetoplastea encode RNA-interacting proteins, with a potential to fine-tune the expression of multiple genes, thus challenging the perception of cis-splicing in these protists as a mere evolutionary relic. We suggest that there is a selective pressure to retain cis-splicing in trypanosomatids and that this is likely associated with overall control of mRNA processing. Our study provides new insights into the evolution of introns and, consequently, the regulation of gene expression in eukaryotes.

• Klíčová slova
• Introns, Kinetoplastea, Poly(A) polymerase, RNA helicase, RNA-binding protein, Splicing, Trypanosomatidae,
• MeSH
• fylogeneze MeSH
• introny * genetika   MeSH
• Kinetoplastida genetika   MeSH
• molekulární evoluce MeSH
• protozoální geny genetika   MeSH
• protozoální proteiny genetika   MeSH
• trans-splicing * genetika   MeSH
• Trypanosomatina genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální proteiny MeSH

The knowledge of cell biology of a eukaryotic group is essential for correct interpretation of ecological and molecular data. Although diplonemid protists are one of the most species-rich lineages of marine eukaryotes, only very fragmentary information is available about the cellular architecture of this taxonomically diverse group. Here, a large serial block-face scanning electron microscopy data set complemented with light and fluorescence microscopy allowed the first detailed three-dimensional reconstruction of a diplonemid species. We describe numerous previously unknown peculiarities of the cellular architecture and cell division characteristic for diplonemid flagellates, and illustrate the obtained results with multiple three-dimensional models, comprehensible for non-specialists in protist ultrastructure.

• Klíčová slova
• 3-dimensional reconstruction, Euglenozoa, SBF-SEM, cell division, diplonemid, ultrastructure,
• MeSH
• Eukaryota * MeSH
• mikroskopie elektronová rastrovací MeSH
• organely MeSH
• zobrazování trojrozměrné * metody   MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• Euglenozoa * klasifikace   cytologie   genetika   MeSH
• Eukaryota cytologie   genetika   MeSH
• mitochondriální ribozomy * metabolismus   MeSH
• ribozomální proteiny metabolismus   MeSH
• RNA ribozomální metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ribozomální proteiny MeSH
• RNA ribozomální MeSH

The β-propeller protein Sec13 plays roles in at least three distinct processes by virtue of being a component of the COPII endoplasmic reticulum export vesicle coat, the nuclear pore complex (NPC) and the Seh1-associated (SEA)/GATOR nutrient-sensing complex. This suggests that regulatory mechanisms coordinating these cellular activities may operate via Sec13. The NPC, COPII and SEA/GATOR are all ancient features of eukaryotic cells, and in the vast majority of eukaryotes, a single Sec13 gene is present. Here we report that the Euglenozoa, a lineage encompassing the diplonemid, kinetoplastid and euglenid protists, possess two Sec13 paralogues. Furthermore, based on protein interactions and localization studies we show that in diplonemids Sec13 functions are divided between the Sec13a and Sec13b paralogues. Specifically, Sec13a interacts with COPII and the NPC, while Sec13b interacts with Sec16 and components of the SEA/GATOR complex. We infer that euglenozoan Sec13a is responsible for NPC functions and canonical anterograde transport activities while Sec13b acts within nutrient and autophagy-related pathways, indicating a fundamentally distinct organization of coatomer complexes in euglenozoan flagellates.

• Klíčová slova
• Diplonema, SEA/GATOR complex, coatomer, membrane trafficking, nuclear pore complex, paralogue expansion,
• MeSH
• buněčná diferenciace MeSH
• Euglenozoa * MeSH
• Eukaryota * MeSH
• eukaryotické buňky MeSH
• jaderný pór MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH