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Essential PBP1-associated proteins of Trypanosoma brucei

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap, LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

TISSUE(S): Cell Culture

DISEASE(S): Trypanosomiasis

SUBMITTER: Thomas Ruppert

LAB HEAD: Thomas Ruppert

PROVIDER: PXD018576 | Pride | 2020-07-14

REPOSITORIES: Pride

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Project description:Variant Surface Glycoproteins (VSG) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. This VSG is a super-abundant virulence factor that is subject to post-transcriptional gene expression controls mediated via the VSG 3’-untranslated region (3’-UTR). To identify positive VSG regulators in bloodstream form cells, we used prior genome-scale screening data to prioritise mRNA binding protein (mRBPs) knockdowns that phenocopy VSG mRNA knockdown, displaying both loss-of-fitness and pre-cytokinesis accumulation. The top three candidate VSG regulators were CFB2 (cyclin F-box protein 2, Tb927.1.4650), MKT1 (Tb927.6.4770) and PBP1 (polyadenylate binding-protein binding-protein, Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate with each other. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify expression profiles following depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of a VSG 3’-untranslated region (3’-UTR). CFB2 knockdown also triggered depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered depletion of CFB2, MKT1, and other components of the PBP1-complex. Finally, all three knockdowns triggered depletion of cytokinesis initiation factors, consistent with the cytokinesis defect that was confirmed here for all three knockdowns. Thus, genome-scale knockdown datasets facilitate the triage and prioritisation of candidate regulators. Quantitative proteomic analysis confirms 3’-UTR dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis.

			Action	DRS
	15-263_msms.txt	Txt
	15-263_proteinGroups.txt	Txt
	E25D070-18-203-01.raw	Raw
	E25D070-18-203-02.raw	Raw
	E25D070-18-203-03.raw	Raw

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Essential PBP1-associated proteins of Trypanosoma brucei

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