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Probing the trypanosome nuclear pore by TurboID proximity labelling – Part2

ABSTRACT: Previous BioID experiments targeting the nucleoporin (NUP) NUP158 (PMID: 36410438; PXD031245), as well as NUP110, NUP76 and NUP96 (PMID: 39206942; PXD047268) indicated that proximity labelling delivers highly specific interactome data in the confined localisation of the nuclear pore, with a labelling radius well below the size of the nuclear pore. The resulting proximity data allowed to assign NUPs and nuclear transport factors to specific subregions of the pore. Here we extended this approach to target NUP75 and transport factors MEX67 and Ran.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Trypanosoma Brucei

TISSUE(S): Permanent Cell Line Cell

DISEASE(S): Trypanosomiasis

SUBMITTER: Martin Zoltner

LAB HEAD: Dr. Martin Zoltner

PROVIDER: PXD055934 | Pride | 2024-09-20

REPOSITORIES: Pride

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			Action	DRS
	MEX67.combined.txt.rar	Txt
	MEX67_1.raw	Raw
	MEX67_2.raw	Raw
	MEX67_3.raw	Raw
	NUP75C_1.raw	Raw

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Proximity labelling and cryomill affinity purification compared for Trypanosoma brucei proteins with unconfined, semi-confined and confined localisation

Project description:Identification of protein-protein interactions is a major contributor for understanding protein function and elucidating molecular and cellular mechanisms. Two strategies are currently popular for identification of protein interaction partners directly from the cellular environment. Affinity purification (pulldown) isolates the protein of interest with the aid of a matrix that specifically captures the target and potential partners. In BioID, the protein of interest is fused to biotin ligase facilitating proximity biotinylation and biotinylated proteins are isolated under stringent conditions with streptavidin. Whilst clear that these two methods can provide insight, it is also apparent that they can reveal distinct interactomes, but the basis for these differences is less obvious. We compare these methods using four different trypanosome proteins as baits: the cytoplasmic poly(A) binding proteins PABP1 and PABP2, mRNA export receptor MEX67, that shuttles between the nucleus and the cytoplasm with predominant localisation at the nuclear pores, and the nucleoporin NUP158.

2022-11-30 | PXD031245 | Pride

Proteins associated wth Trypanosoma brucei HNRNPF/H

Project description:We aimed to find proteins associated with HNRNPH in bloodstream-form _Trypanosoma brucei_. We integrated a sequence encoding a cleavable TAP (Tandem Affinity Purification) tag into the genome upstream of, and in frame with, one _HNRNPH_ gene. The other allele was deleted. TAP-HNRNPH was purifed from triplicate lysates as shown under "Sample processing protocol". Sample processing protocol (30-5000chracters) The TAP-HNRNPH was bound to IgG beads, then released with Tobacco Etch Virus (TEV) protease. TAP-CFP served as the negative control. The His-tagged TEV protease was then removed using Ni-NTA-magnetic beads. Eluted proteins were separated on a 1.5 mm NuPAGE™ Novex™ 4-12 % Bis-Tris protein gel. For details see doi: 10.1371/journal.pone.0258903

2022-10-15 | PXD037289 | Pride

Essential PBP1-associated proteins of Trypanosoma brucei

Project description:Control of gene expression in kinetoplastids depends heavily on RNA-binding proteins that influence mRNA decay and translation. We previously showed that MKT1 interacts with PBP1, which in turn recruits LSM12 and poly(A) binding protein. MKT1 is recruited to mRNA by sequence-specific RNA-binding proteins, resulting in stabilisation of mRNA. We here show that PBP1, LSM12 and an additional 117-residue protein, XAC1 (Tb927.7.2780), are present in complexes that contain either MKT1 or MKT1L (Tb927.10.1490). All five proteins are present predominantly in the complexes, and there was evidence for a minor subset of complexes that contained both MKT1 and MKT1L. MKT1 appeared to be associated with many mRNAs, with the exception of those encoding ribosomal proteins. XAC1-containing complexes reproducibly contained RNAbinding proteins that were previously found associated with MKT1. In addition, however, XAC1- or MKT1- containing complexes specifically recruit one of the six translation initiation complexes, EIF4E6-EIF4G5; and yeast 2-hybrid assay results indicated that MKT1 interacts with EIF4G5. The C-terminus of MKT1L resembles MKT1: it contains MKT1 domains and a PIN domain that is probably not active as an endonuclease. MKT1L, however, also has an N-terminal extension with regions of low-complexity. Although MKT1L depletion inhibited cell proliferation, we found no evidence for specific interactions with RNA-binding proteins or mRNA. Deletion of the N-terminal extension, however, enabled MKT1L to interact with EIF4E6. We speculate that MKT1L may either enhance or inhibit the functions of MKT1-containing complexes.

2020-07-14 | PXD018576 | Pride

Probing the trypanosome nuclear pore by TurboID proximity labelling

Project description:Previous BioID experiments targeting the nucleoporin (NUP) NUP158 and the nuclear transport factor MEX67 indicated (PMID: 36410438; PXD031245) that proximity labelling delivers highly specific interactome data in the confined localisation of the nuclear pore, with a labelling radius well below the size of the nuclear pore. Here we extended this approach to target NUPs NUP110, NUP76 and NUP96 with a TurboID-HA tandem tag fused to both, the N and C-terminus of each respective target protein. While the central NUP96 biotinylated most nuclear pore proteins with the marked exception of the nuclear basket, NUP158, NUP110 and NUP76 caused biotinylation of specific sub-complexes of the pore and Mex67 labelled NUPs lining the pore channel.

2023-12-04 | PXD047268 | Pride

Exploring the vault interactome in the divergent eukaryote Trypanosoma brucei

Project description:Many, but not all Eukaryotes have protein-enclosed compartments called vaults. Vaults are composed of multiple copies of the major vault protein (MVP), symmetrically assembled into a basket like shell. Whilst there is some evidence that these compartments are engaging in mRNA maturation and intriguing structural information of the vault shell is available, the function of vaults remains largely elusive. Here, we set out to explore the vault interactome in the early branching eukaryote Trypanosoma brucei employing a combination of affinity capture and TurboID proximity labelling.

2024-12-12 | PXD056004 | Pride

TEF-seq/NET-seq applied to factors Rpb3, Paf1, Spt6, Spt16, Cet1, Pcf11, Ssu72, Set2

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2021-01-18 | E-MTAB-4568 | biostudies-arrayexpress

A novel essential RNA-binding protein complex in Trypanosoma brucei

Project description:ZC3H5 is an essential cytoplasmic trypanosome protein with a single Cx7Cx5Cx3H zinc finger domain. We here show that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500 and Tb927.7.3040. ZC3H5 interacts directly with Tb927.11.4900, which in turn interacts with Tb927.7.3040. Tb927.11.4900 has a circularly permuted GTPase domain, which is required for the Tb927.7.3040 interaction. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which were enriched in the motif (U/A)UAG(U/A). Tethering of ZC3H5, or other complex components, to a reporter repressed its expression. However, depletion of ZC3H5 in vivo did not increase the abundance of ZC3H5-bound mRNAs; instead, counter-intuitively, there were very minor decreases in a few targets. This was accompanied by marked increases in the abundance of very stable mRNAs encoding ribosomal proteins. Depletion also resulted in an increase in monosomes at the expense of large polysomes, and appearance of a "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex is implicated in quality control during the translation of sub-optimal open reading frames, and that its assembly is regulated by GTP hydrolysis and GTP-GDP exchange.

2020-09-23 | PXD019073 | Pride

A unique mRNA decapping complex in trypanosomes

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2023-06-14 | PXD038550 | Pride

MX2 forms nucleoporin-comprising cytoplasmic biomolecular condensates that lure viral capsids

Project description:Human myxovirus resistance 2 (MX2) can restrict HIV-1 and herpesviruses at a post-entry step through a process requiring an interaction between MX2 and the viral capsids. The involvement of other host cell factors, however, remains poorly understood. Here, we mapped the proximity interactome of MX2, revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and anti-herpes simplex virus 1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region and MX2 dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates, preventing nuclear entry of their viral genomes. Thus, MX2 forms cytoplasmic condensates that likely act as nuclear pore decoys, trapping capsids and inducing premature viral genome release to interfere with nuclear targeting of HIV-1 and HSV-1.

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