Biology subjects

Gabiatti, B. P.

Publications and source records attributed to Gabiatti, B. P..

3 recordsLinked to original sources

bioRxiv2024

An updated map of the trypanosome nuclear pore and its associated proteins

Nuclear export of mRNAs requires loading the mRNP to the transporter Mex67/Mtr2 in the nucleoplasm, controlled access to the pore by the basket-localized TREX2 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 TREX2 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. The resulting availability of reference proteins for basket, inner ring and cytoplasmic site allowed mapping of all 75 trypanosome proteins with known nuclear pore localisation to a sub-region of the pore based on mass spectrometry data from proximity labelling. This approach defined many further asymmetrically localised nuclear pore components. At the nuclear site, we identified several trypanosome-unique proteins, for instance the FG-NUPs NUP64/NUP98, but also proteins with structural homology to TREX-2 components. We mapped the components of the Ran-based nuclear export system and confirm the absence of a Dbp5 homologue. Lastly, we demonstrate, by deploying an auxin degron system, that NUP76 holds an essential role in mRNA export consistent with a 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 fort 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.

cell biology↗

bioRxiv2024

Intron-loss in Kinetoplastea correlates with a non-functional EJC and loss of NMD factors

In metazoans, mRNA quality is tightly monitored from transcription to translation. A key role lies with the exon junction complex (EJC) that is placed upstream of the exon-exon junction after splicing. The EJC 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 non-sense mediated decay (NMD, UPF1/2/3). The protozoan parasite Trypanosoma brucei encodes only two genes with introns, but all mRNAs are processed by trans-splicing. The presence of the three core EJC proteins and a potential BTZ homologue (Rbp25) in trypanosomes has been suggested as an adaptation 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 and does not affect processing, export or stability of the two cis-spliced mRNAs (ii) eIF4AIII localization is mostly nucleolar, with a minor proportion in the nucleoplasm and cytoplasm, while Magoh and Y14 are nucleolar and nucleoplasmic but excluded from the cytoplasm (iii) eIF4AIII associates with nucleolar proteins (including NOM1/Sgd1p) 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 the nucleolar proteins. Our data argue against the presence of a functional EJC in trypanosomes, but rather 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. We found that depletion of UPF1 causes only a minor reduction in growth and detailed phylogenetic analyses show several independent losses of UPF1 and UPF2, as well as total loss of UPF3 in the Kinetoplastida group. The data indicate that UPF1-dependent NMD is not essential in trypanosomes consistent with the observation that the cells tolerate the presence of mRNAs with allele-specific, endogenous PTCs (premature termination codons). Altogether, our comprehensive analyses of conserved components provide no evidence for a canonical EJC or NMD pathway in (almost) intron-less trypanosomatids.

biochemistry↗

bioRxiv2023

Beyond BioID: Streptavidin outcompetes antibody fluorescence signals in protein localization and readily visualises targets evading immunofluorescence detection.

Immunofluorescence is a common method to localise proteins within their cellular context via fluorophore labelled antibodies and for some applications without alternative. However, some protein targets evade detection due to low protein abundance or accessibility issues. In addition, some imaging methods require a massive reduction in antigen density thus impeding detection of even medium-abundant proteins. Here, we show that the fusion of the target protein to TurboID, a biotin ligase labelling lysine residues in close proximity, and subsequent detection of biotinylation by fluorescent streptavidin offers an "all in one" solution to the above-mentioned restrictions. For a wide range of target proteins tested, the streptavidin signal was significantly stronger than an antibody signal, markedly improving the imaging sensitivity in expansion microscopy and correlative light and electron microscopy, with no loss in resolution. 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 fail to label proteins in these environments. When TurboID is used in tandem with an HA epitope tag, co-probing with streptavidin and anti-HA can be used to map antibody- accessibility to certain cellular regions. As a proof of principle, we mapped antibody access to all trypanosome nuclear pore proteins (NUPs) and found restricted antibody labelling of all FG NUPs of the central channel that are known to be phase-separated, while most non-FG Nups could be labelled. Lastly, we show that streptavidin imaging can resolve dynamic, temporally and spatially distinct sub-complexes and, in specific cases, reveal 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, can provide information on protein interactions and biophysical environment.

cell biology↗