The nuclear envelope is subtended in most eukaryotes by a proteinaceous lamina, a network that has been recognised since the 1950s. Originally considered as a simple structural support, it is now clear that the lamina can be highly dynamic and participates in a multitude of functions, including transcriptional and epigenetic regulation, definition of chromatin domains, genome stability and the positioning of nuclear pore complexes. The major protein components of the lamina in metazoans are ~60 kDa lamins, which assemble to form fibres and a network and are regulated by multiple mechanisms. Despite a broad taxonomic distribution beyond Metazoa, lamins are not universal and, in at least three major lineages, are absent, specifically fungi, plants and kinetoplastid protists. The latter two possess lineage-specific lamin analogues, the CRWN and NUP-1/NUP-2 proteins, respectively. Here we discuss and compare the kinetoplastid and plant lamina, their origins, components and functions and spectacular examples of convergent evolution.

• Klíčová slova
• CRWN proteins, Eukaryogenesis, Evolution, Gene expression, Lamina, Nuclear pore complex, Nucleus, Plants, Trypanosomes,
• MeSH
• jaderná lamina * metabolismus   MeSH
• laminy * metabolismus   genetika   MeSH
• lidé MeSH
• rostliny metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• laminy * MeSH

The exon junction complex (EJC) is a key player in metazoan mRNA quality control and is placed upstream of the exon-exon junction after splicing. Its inner core is composed of Magoh, Y14, eIF4AIII and BTZ and the outer core of proteins involved in mRNA splicing (CWC22), export (Yra1), translation (PYM) and nonsense mediated decay (NMD, UPF1/2/3). Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested to adapt of the EJC function to mark trans-spliced mRNAs. We analysed trypanosome EJC components and noticed major differences between eIF4AIII and Magoh/Y14: (i) whilst eIF4AIII is essential, knocking out both Magoh and Y14 elicits only a mild growth phenotype (ii) eIF4AIII localization is mostly nucleolar, while Magoh and Y14 are nucleolar and nucleoplasmic but excluded from the cytoplasm (iii) eIF4AIII associates with nucleolar proteins and the splicing factor CWC22, but not with Y14 or Magoh, while Magoh and Y14 associate with each other, but not with eIF4AIII, CWC22 or nucleolar proteins. Our data argue against the presence of a functional EJC in trypanosomes, but indicate that eIF4AIII adopted non-EJC related, essential functions, while Magoh and Y14 became redundant. Trypanosomes also possess homologues to the NMD proteins UPF1 and UPF2. Depletion of UPF1 causes only a minor reduction in growth and phylogenetic analyses show several independent losses of UPF1 and UPF2, as well as complete loss of UPF3 in the Kinetoplastida group, indicating that UPF1-dependent NMD is not essential. Regardless, we demonstrate that UPF1 depletion restores the mRNA levels of a PTC reporter. Altogether, we show that the almost intron-less trypanosomes are in the process of losing the canonical EJC/NMD pathways: Y14 and Magoh have become redundant and the still-functional UPF1-dependent NMD pathway is not essential.

• MeSH
• eukaryotický iniciační faktor 4A metabolismus   genetika   MeSH
• exony genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• nonsense mediated mRNA decay * MeSH
• protozoální proteiny * metabolismus   genetika   MeSH
• RNA-helikasy * metabolismus   genetika   MeSH
• sestřih RNA MeSH
• Trypanosoma brucei brucei * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 4A MeSH
• messenger RNA MeSH
• protozoální proteiny * MeSH
• RNA-helikasy * MeSH

Nuclear export of mRNAs requires loading the mRNP to the transporter Mex67/Mtr2 in the nucleoplasm, controlled access to the pore by the basket-localised TREX-2 complex and mRNA release at the cytoplasmic site by the DEAD-box RNA helicase Dbp5. Asymmetric localisation of nucleoporins (NUPs) and transport components as well as the ATP dependency of Dbp5 ensure unidirectionality of transport. Trypanosomes possess homologues of the mRNA transporter Mex67/Mtr2, but not of TREX-2 or Dbp5. Instead, nuclear export is likely fuelled by the GTP/GDP gradient created by the Ran GTPase. However, it remains unclear, how directionality is achieved since the current model of the trypanosomatid pore is mostly symmetric. We have revisited the architecture of the trypanosome nuclear pore complex using a novel combination of expansion microscopy, proximity labelling and streptavidin imaging. We could confidently assign the NUP76 complex, a known Mex67 interaction platform, to the cytoplasmic site of the pore and the NUP64/NUP98/NUP75 complex to the nuclear site. Having defined markers for both sites of the pore, we set out to map all 75 trypanosome proteins with known nuclear pore localisation to a subregion of the pore using mass spectrometry data from proximity labelling. This approach defined several further proteins with a specific localisation to the nuclear site of the pore, including proteins with predicted structural homology to TREX-2 components. We mapped the components of the Ran-based mRNA export system to the nuclear site (RanBPL), the cytoplasmic site (RanGAP, RanBP1) or both (Ran, MEX67). Lastly, we demonstrate, by deploying an auxin degron system, that NUP76 holds an essential role in mRNA export consistent with a possible functional orthology to NUP82/88. Altogether, the combination of proximity labelling with expansion microscopy revealed an asymmetric architecture of the trypanosome nuclear pore supporting inherent roles for directed transport. Our approach delivered novel nuclear pore associated components inclusive positional information, which can now be interrogated for functional roles to explore trypanosome-specific adaptions of the nuclear basket, export control, and mRNP remodelling.

• MeSH
• aktivní transport - buněčné jádro MeSH
• buněčné jádro metabolismus   MeSH
• jaderný pór * metabolismus   ultrastruktura   MeSH
• komplex proteinů jaderného póru metabolismus   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• nukleocytoplazmatické transportní proteiny metabolismus   MeSH
• proteiny vázající RNA metabolismus   MeSH
• protozoální proteiny metabolismus   genetika   MeSH
• ribonukleoproteiny MeSH
• transport RNA MeSH
• Trypanosoma brucei brucei * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• komplex proteinů jaderného póru MeSH
• messenger ribonucleoprotein MeSH Prohlížeč
• messenger RNA * MeSH
• nukleocytoplazmatické transportní proteiny MeSH
• proteiny vázající RNA MeSH
• protozoální proteiny MeSH
• ribonukleoproteiny 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