An updated map of the trypanosome nuclear pore and its associated proteins DOI Creative Commons
Bernardo Gabiatti, Johanna Odenwald, Silke Braune

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Окт. 6, 2024

ABSTRACT Nuclear export of mRNAs requires loading the mRNP to transporter Mex67/Mtr2 in nucleoplasm, controlled access pore by basket-localized TREX2 complex and mRNA release at cytoplasmic site DEAD-box RNA helicase Dbp5. Asymmetric localisation nucleoporins (NUPs) transport components as well ATP dependency Dbp5 ensure unidirectionality transport. Trypanosomes possess homologues Mex67/Mtr2, but not or Instead, nuclear is likely fuelled GTP/GDP gradient created Ran GTPase. However, it remains unclear, how directionality achieved since current model trypanosomatid mostly symmetric. We have revisited architecture trypanosome using a novel combination expansion microscopy, proximity labelling streptavidin imaging. could confidently assign NUP76 complex, known Mex67 interaction platform, pore. The resulting availability reference proteins for basket, inner ring allowed mapping all 75 with sub-region based on mass spectrometry data from labelling. This approach defined many further asymmetrically localised components. At site, we identified several trypanosome-unique proteins, instance FG-NUPs NUP64/NUP98, also structural homology TREX-2 mapped Ran-based system confirm absence homologue. Lastly, demonstrate, deploying an auxin degron system, that holds essential role consistent functional orthology NUP82/88. Altogether, microscopy revealed asymmetric supporting inherent roles fort directed Our delivered associated inclusive positional information, which can now be interrogated explore specific adaptions control remodelling.

Язык: Английский

Detailed characterisation of the trypanosome nuclear pore architecture reveals conserved asymmetrical functional hubs that drive mRNA export DOI Creative Commons
Bernardo Gabiatti,

Johanna Krenzer,

Silke Braune

и другие.

PLoS Biology, Год журнала: 2025, Номер 23(2), С. e3003024 - e3003024

Опубликована: Фев. 3, 2025

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

Язык: Английский

Процитировано

1
Multifunctional roles of Sec13 paralogues in the euglenozoan Trypanosoma brucei DOI Creative Commons
Mohamed Sharif,

Lydia Greenberg,

James D. Bangs

и другие.

Open Biology, Год журнала: 2025, Номер 15(2)

Опубликована: Фев. 1, 2025

Secretory cargos are exported from the ER via COPII-coated vesicles that have an inner matrix of Sec23/Sec24 heterotetramers and outer cage Sec13/Sec31 heterotetramers. In addition to COPII, Sec13 is part nuclear pore complex (NPC) regulatory SEA/GATOR in eukaryotes, which typically one orthologue. The kinetoplastid parasite Trypanosoma brucei has two paralogues: TbSec13.1, accepted component both COPII NPC, TbSec13.2. Little known about TbSec13.2, but others proposed it, its orthologue distantly related diplonemid Paradiplonema papillatum , operate exclusively complex, this represents evolutionary diversification function unique euglenozoan protists. Using RNAi silencing trypanosomes, we show TbSec13s essential. Knockdown each dramatically equally delays transport GPI-anchored secretory cargo, indicating roles for COPII-mediated trafficking ER. Immunofluorescence proximity labelling studies confirm TbSec13.1 TbSec13.2 co-localize with TbSec24.1 exit sites, thus functional components machinery. Our findings indicate not restricted trypanosomes.

Язык: Английский

Процитировано

0
Trypanosomes lack a canonical EJC but possess an UPF1 dependent NMD-like pathway DOI Creative Commons
Bernardo Gabiatti,

Eden Ribeiro Freire,

Johanna Odenwald

и другие.

PLoS ONE, Год журнала: 2025, Номер 20(3), С. e0315659 - e0315659

Опубликована: Март 7, 2025

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

Язык: Английский

Процитировано

0
A comprehensive toolkit for protein localization and functional analysis in trypanosomatids DOI Creative Commons

Athina Paterou,

Julia Sáez Conde,

Jiří Týč

и другие.

Open Biology, Год журнала: 2025, Номер 15(4)

Опубликована: Апрель 1, 2025

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

Язык: Английский

Процитировано

0
An updated map of the trypanosome nuclear pore and its associated proteins DOI Creative Commons
Bernardo Gabiatti, Johanna Odenwald, Silke Braune

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Окт. 6, 2024

ABSTRACT Nuclear export of mRNAs requires loading the mRNP to transporter Mex67/Mtr2 in nucleoplasm, controlled access pore by basket-localized TREX2 complex and mRNA release at cytoplasmic site DEAD-box RNA helicase Dbp5. Asymmetric localisation nucleoporins (NUPs) transport components as well ATP dependency Dbp5 ensure unidirectionality transport. Trypanosomes possess homologues Mex67/Mtr2, but not or Instead, nuclear is likely fuelled GTP/GDP gradient created Ran GTPase. However, it remains unclear, how directionality achieved since current model trypanosomatid mostly symmetric. We have revisited architecture trypanosome using a novel combination expansion microscopy, proximity labelling streptavidin imaging. could confidently assign NUP76 complex, known Mex67 interaction platform, pore. The resulting availability reference proteins for basket, inner ring allowed mapping all 75 with sub-region based on mass spectrometry data from labelling. This approach defined many further asymmetrically localised components. At site, we identified several trypanosome-unique proteins, instance FG-NUPs NUP64/NUP98, also structural homology TREX-2 mapped Ran-based system confirm absence homologue. Lastly, demonstrate, deploying an auxin degron system, that holds essential role consistent functional orthology NUP82/88. Altogether, microscopy revealed asymmetric supporting inherent roles fort directed Our delivered associated inclusive positional information, which can now be interrogated explore specific adaptions control remodelling.

Язык: Английский

Процитировано

0