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
• jaderný pór * metabolismus   MeSH
• komplex proteinů jaderného póru * metabolismus   genetika   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• nukleocytoplazmatické transportní proteiny metabolismus   genetika   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 RNA * MeSH
• nukleocytoplazmatické transportní proteiny MeSH
• protozoální proteiny * MeSH

Immunofluorescence localises proteins via fluorophore-labelled antibodies. However, some proteins evade detection due to antibody-accessibility issues or because they are naturally low abundant or antigen density is reduced by the imaging method. Here, we show that the fusion of the target protein to the biotin ligase TurboID and subsequent detection of biotinylation by fluorescent streptavidin offers an 'all in one' solution to these restrictions. For all proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the sensitivity of expansion microscopy and correlative light and electron microscopy. Importantly, proteins within phase-separated regions, such as the central channel of the nuclear pores, the nucleolus, or RNA granules, were readily detected with streptavidin, while most antibodies failed. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can map antibody-accessibility and we created such a map for the trypanosome nuclear pore. Lastly, we show that streptavidin imaging resolves dynamic, temporally, and spatially distinct sub-complexes and, in specific cases, reveals a history of dynamic protein interaction. In conclusion, streptavidin imaging has major advantages for the detection of lowly abundant or inaccessible proteins and in addition, provides information on protein interactions and biophysical environment.

• Klíčová slova
• BioID, Trypanosoma brucei, TurboID, cell biology, human, nuclear pore, phase separation, streptavidin imaging,
• MeSH
• biotinylace MeSH
• fluorescenční mikroskopie metody   MeSH
• lidé MeSH
• protilátky metabolismus   MeSH
• protozoální proteiny imunologie   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   genetika   imunologie   MeSH
• streptavidin * chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• protilátky MeSH
• protozoální proteiny MeSH
• rekombinantní fúzní proteiny MeSH
• streptavidin * MeSH

Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.

• MeSH
• endoribonukleasy * metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• RNA čepičky * genetika   metabolismus   MeSH
• stabilita RNA MeSH
• Trypanosoma * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endoribonukleasy * MeSH
• messenger RNA MeSH
• mRNA decapping enzymes MeSH Prohlížeč
• proteiny vázající RNA MeSH
• RNA čepičky * MeSH