Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G 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 remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, 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 essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes

Impact of inherent biases built into proteomic techniques:Proximity labeling and affinity capture compared

The characterization of protein–protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches

A unique mRNA decapping complex in trypanosomes.

Acknowledgements: We are grateful to the FingerPrints proteomics facility at the University of Dundee and the OMICS Proteomics BIOCEV core facility for excellent technical service. We also thank the Mass Spectrometry facility P02-004 at Fiocruz-Carlos Chagas Institute and the Core Facility for Crystallography and Biophysics (University of Warsaw) for valuable support. We thank Andrea Reichert for her work on the phenotypic analysis of ALPH1ΔN/– cells, Nico Ankenbrand for technical assistance and Tim Krüger for help with imaging (all University of Würzburg).Funder: CAPES; doi: https://doi.org/10.13039/501100002322Funder: Foundation for Polish Science; doi: https://doi.org/10.13039/501100001870Funder: Fiocruz Technological Platform programFunder: University library WürzburgRemoval of the mRNA 5' cap 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. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts

Functional Genomic Characterization of mRNAs Associated with TcPUF6, a Pumilio-like Protein from Trypanosoma cruzi*S⃞

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle

Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei.

Peer reviewed: TrueAcknowledgements: The authors are grateful to Nancy Standart for critical review of the manuscript. They also acknowledge the substantial support from the FIOCRUZ Technical Platform program through the Platforms for the high throughput SOLID sequencing and the facility for Protein Purification and Characterization and Proteomics at the Carlos Chagas Institute (Fiocruz Parana), as well as the DNA Sequencing and Electron Microscopy Platforms at the Aggeu Magalhaes Institute (Fiocruz Pernambuco).Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G 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 remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, 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 essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes

Image4_Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei.PDF

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G 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 remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, 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 essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.</p

Image3_Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei.PDF

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G 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 remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, 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 essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.</p

Image1_Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei.PDF

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G 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 remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, 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 essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.</p