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Interactome of the two T. brucei Poly(A) binding protein paralogs PABP1and PABP2

ABSTRACT: Poly(A) binding proteins (PABPs) regulate mRNA fate by controlling mRNA stability and translation through interactions with both the poly(A) tail and eIF4F complex. Here we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

TISSUE(S): Cell Line Cell

DISEASE(S): Trypanosomiasis

SUBMITTER: Martin Zoltner

LAB HEAD: Mark C. Field

PROVIDER: PXD008839 | Pride | 2018-07-16

REPOSITORIES: pride

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			Action	DRS
	Experiment10_PAPB2eYFP_highsalt.raw	Raw
	Experiment11_PABP2eYFP_lowsalt.raw	Raw
	Experiment12_PABP2eYFP_lowsalt.raw	Raw
	Experiment1_PABP1eYFP_highsalt.raw	Raw
	Experiment2_PABP1eYFP_highsalt.raw	Raw

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Publications

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA.

Zoltner Martin M Krienitz Nina N Field Mark C MC Kramer Susanne S

PLoS neglected tropical diseases 20180724 7

Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation i ...[more]

PMID: 30040867

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Immunoisolation of the trypanosome decapping enzyme ALPH1 delivers a potential subcomplex

Project description:Removal of mRNA 5’ caps 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. The enzyme is composed of a catalytic domain flanked by C-terminal and N-terminal extensions. In our related deposition PXD038550 we analysed the ALPH1 interactome by BioID proximity labelling for the full length protein and truncated versions in order to assign domain specific interactions. We showed that Trypanosoma brucei ALPH1 acts in a complex composed of the trypanosome XRN1 ortholog XRNA and four proteins that are unique to Kinetoplastida. The interactome was validated by reverse experiments targeting T. brucei and T. cruzi XRNA by affinity capture and, additionally, the ALPH1 interacting CMGC-family kinase by BioID. Here we carried out affinity capture with T. brucei and T. cruzi ALPH1, which delivers a potential sub-complex missing the C-terminal interactor XRNA.

2023-06-14 | PXD042322 | Pride

A unique mRNA decapping complex in trypanosomes

Project description:Removal of mRNA 5’ caps 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. The enzyme is composed of a catalytic domain flanked by C-terminal and N-terminal extensions. Here we analyse the ALPH1 interactome by BioID proximity labelling for the full length protein and truncated versions in order to assign domain specific interactions. We show that Trypanosoma brucei ALPH1 acts in a complex composed of the trypanosome XRN1 ortholog XRNA and four proteins that are unique to Kinetoplastida.The interactome is validated by reverse experiments targeting T. brucei and T. cruzi XRNA by affinity capture and, additionally, the ALPH1 interacting CMGC-family kinase by BioID.

2023-06-14 | PXD038550 | 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

Mass spectrometry analysis of four translation initiation proteins, EIF4E3, EIF4E4, EIF4G3 and EIF4G4 in Trypanosoma brucei

Project description:Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, for example in protein synthesis there are multiple eIF4F-like complexes. The eukaryotic eIF4F complex, formed by the eIF4E, eIF4G and eIF4A subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions have yet to be properly identified. Here, to define the interactome associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the two sets of tagged eIF4E and eIF4G subunits. A number of different protein partners, including RBPs and RNA helicases, were found to specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases.EIF4E3/EIF4G4 are known to be essential for viability and to better define their role.

2023-03-10 | PXD033857 | 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

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

Characterisation of cullin E3 ubiquitin ligases in trypanosomes

Project description:Trypanosomes have seven cullin paralogs, the majority lineage-specific. Six of those cullins (TbCul-A, TbCul-C, TbCul-D TbCul-E, TbCul-F and TbCul-G) were endogenously 3xHA-epitope tagged at the C-terminus. We used affinity capture/mass spectrometry to determine the composition of these cullin complexes. TbCul-A and TbCul-E function were further studied by analysis of proteome changes upon upon RNAi.

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Project description:UV-crosslinking and immunoprecipitation (CLIP) combined with high-throughput sequencing was previously used to generate transcriptome-wide binding maps of several RNA-binding proteins9-12. However, since identification of binding sites relied on the analysis of overlapping sequence clusters, distances of less than 30 nucleotides were not resolved. An additional disadvantage of CLIP is the requirement of reverse transcription to pass over residual amino acids that remain covalently attached to the RNA at the crosslink site. Primer extension assays have shown that the vast majority of cDNAs prematurely truncate immediately before the 'crosslink nucleotide'13. Here, we exploited this apparent limitation to achieve single nucleotide resolution by capturing these truncated cDNAs through the introduction of a second adapter after reverse transcription via self-circularization. In order to quantify cDNA molecules that truncate at the same nucleotide, we added a random barcode to the DNA adapter. This allowed us to discriminate between unique cDNA products and PCR duplicates . Taken together, individual-nucleotide resolution CLIP (iCLIP) enables precise mapping of protein-RNA interactions in intact cells.

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