African trypanosomes are medically important parasites that cause sleeping sickness in humans and nagana in animals. In addition to their pathogenic role, they have emerged as valuable model organisms for studying fundamental biological processes. Protein tagging is a powerful tool for investigating protein localization and function. In a previous study, we developed two plasmids for rapid and reproducible polymerase chain reaction-based protein tagging in trypanosomes, which enabled the subcellular mapping of 89% of the trypanosome proteome. However, the limited selection of fluorescent protein tags and selectable markers restricted the flexibility of this approach. Here, we present an extended set of >100 plasmids that incorporate universal primer annealing sequences, enabling protein tagging with a range of fluorescent, biochemical and epitope tags, using five different selection markers. We evaluated the suitability of various fluorescent proteins for live and fixed cell imaging, fluorescent movies, and we demonstrate the use of tagging plasmids encoding tandem epitope tags to support expansion microscopy approaches. We show that this series of plasmids is functional in other trypanosomatid parasites, significantly increasing its value. Finally, we developed a new plasmid for tagging glycosylphosphatidylinositol-anchored proteins. We anticipate that this will be an important toolset for investigating trypanosomatid protein localization and function.

• Klíčová slova
• expansion microscopy, protein tagging, toolkit, trypanosomatid, trypanosome,
• MeSH
• lidé MeSH
• plazmidy genetika   MeSH
• protozoální proteiny * metabolismus   genetika   MeSH
• transport proteinů MeSH
• Trypanosoma brucei brucei metabolismus   genetika   MeSH
• Trypanosomatina * metabolismus   genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protozoální proteiny * 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
• 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