Abstract
Trypanosomatids are parasitic protozoans characterized by several unique structural and metabolic processes that include exquisite mechanisms associated with gene expression and regulation. During the initiation of protein synthesis, for instance, mRNA selection for translation seems to be mediated by different eIF4F-like complexes, which may play a significant role in parasite adaptation to different hosts. In eukaryotes, the heterotrimeric eIF4F complex (formed by eIF4E, eIF4G, and eIF4A) mediates mRNA recognition and ribosome binding and participates in various translation regulatory events. Six eIF4Es and five eIF4Gs have been described in trypanosomatids with several of these forming different eIF4F-like complexes. This has raised questions about their role in differential mRNA translation. Here we have studied further TbEIF4E2, the least known eIF4E homologue from Trypanosoma brucei, and found that it is not associated with an eIF4G homolog. It is, however, associated with mature mRNAs and binds to a histone mRNA stem-loop-binding protein (SLBP), one of two Trypanosoma SLBP homologs (TbSLBP1 and TbSLBP2). TbSLBP1 is more similar to the mammalian counterpart while TbSLBP2 is exclusive to trypanosomatids and related organisms. TbSLBP2 binds to TbEIF4E2 through a conserved central region missing in other SLBP homologs. Both SLBPs, as well as TbEIF4E2, were found to localize to the cytoplasm. TbEIF4E2 and TbSLBP2 are differentially expressed during cell culture, being more abundant in early-log phase, with TbSLBP2 also showing cell-cycle dependent expression. The new data reinforce unique aspects of the trypanosomatid eIF4Es, with the TbEIF4E2–TbSLBP complex possibly having a role in differential selection of mRNAs containing stem-loop structures.
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Acknowledgements
The authors thank Dr. Mark Carrington, from University of Cambridge (UK), and Dr. Steve Kelly, from Department of Plant Sciences, University of Oxford (UK) for providing the SLBP protein sequences from Euglena gracilis. We also thank Dr. Cássia Docena from the Núcleo de Plataformas Tecnológicas (NPT), Instituto Aggeu Magalhães, Fiocruz-PE, for the assistance in obtaining Immunofluorescence images. The anti-BiP and anti-TY antibodies were kind gifts from Jay Bangs and Keith Gull, respectively. This work was supported by a UCLA Stein-Oppenheimer award (D. C.), the National Institutes of Health (Grant numbers AI056034, AI073806, TW009035 to D. C. and N. S., Grant number GM089778 to J. W.), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil, Grant number 401282/2014-7) and Fundação Oswaldo Cruz-Fiocruz (Brazil). Funding was provided by Fogarty International Center (PAR-08-222).
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Supplementary figure 1. Multiple sequence alignment comparing the SLBP1 amino acid sequences from selected trypanosomatid species and the related
B. saltans ortholog. The RBD domain is highlighted by dashes with asterisks indicating conserved residues involved in mRNA stem-loop binding. Alignment indicates conserved regions of SLBP1 from B. saltans (BsSLBP1, GeneDB#: BSAL_11875; NCBI#: CUG87777.1), T. brucei (TbSLBP1, GeneDB#: Tb927.3.1910; NCBI#: XP_XP_843785.1), T. cruzi (TcSLBP1, GeneDB#: TcCLB.430061.9; NCBI#: XP_802584.1), L. major (LmSLBP1, GeneDB#: LmjF.25.1830; NCBI#: XP_001683942.1) and L. braziliensis (LbSLBP1, GeneDB#: LbrM.25.1390; NCBI# XP_001565643.1). Identical amino acids are indicated by black shading. Amino acids defined as similar, by the BLOSUM 62 Matrix, in >50% of the sequences are shaded gray Supplementary material 1 (EPS 2587 KB)
Supplementary figure 2. Multiple sequence alignment comparing the SLBP2 amino acid sequences from selected trypanosomatid species and the related
B. saltans ortholog. The RBD domain is highlighted by dashes with asterisks indicating conserved residues involved in mRNA stem-loop binding. The red box shows the conserved region found in SLBP2 orthologs only from B. saltans (BsSLBP2, GeneDB#: BSAL_85420; Pubmed#: CUG77401.1), T. brucei (TbSLBP2, GeneDB#: Tb927.03.870; Pubmed#: XP_843683.1), T. cruzi (TcSLBP2, GeneDB#: TcCLB.511867.150; Pubmed#: XP_814962.1), L. major (LmSLBP2, GeneDB#: LmjF.25.0920; Pubmed#: XP_001683846.1) and L. braziliensis (LbSLBP2, GeneDB#: LbrM.25.0800; Pubmed# XP_001565585.1). Identical amino acids are indicated by black shading. Amino acids defined as similar, by the BLOSUM 62 Matrix, in >60% of the sequences are shaded gray Supplementary material 2 (EPS 2440 KB)
Supplementary figure 3. Yeast two-hybrid assay confirms lack of interaction between TbEIF4E2 and the five
T. brucei EIF4G homologs. Interactions between TbEIF4E2 and TbEIF4G1 trough G5 homologs was assayed using the yeast two-hybrid assay in the presence of increasing amounts of 3AT, increasing the stringency of the assay. Positive controls were pGADT7-T and pGBKT7-53; the negative controls were the empty vectors Supplementary material 3 (EPS 1252 KB)
Supplementary figure 4. TbSLBP2-PTP construction
. (A) Schematic representation showing the TbSLBP2 cell line construction with integration of the PTP tag into the allele to generate the TbSLBP2wt/PTP cell line (same strategy was used for TbSLBP1wt/PTP cell line). (B) Western blot of extracts from the TbSLBP1wt/PTP and TbSLBP2wt/PTP cell lines using an antibody against the protein A domain of the PTP tag Supplementary material 4 (EPS 1408 KB)
Supplementary figure 5. mRNA levels of TbEIF4E2 and TbSLBP2
. Combined data from the transcriptome of T. brucei shows differential abundance over the cell cycle in procyclic cells for TbEIF4E2 and TbSLBP2 but not TbSLBP1 Supplementary material 5 (EPS 1050 KB)
Supplementary table 1.
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Supplementary table 2.
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Freire, E.R., Moura, D.M.N., Bezerra, M.J.R. et al. Trypanosoma brucei EIF4E2 cap-binding protein binds a homolog of the histone-mRNA stem-loop-binding protein. Curr Genet 64, 821–839 (2018). https://doi.org/10.1007/s00294-017-0795-3
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DOI: https://doi.org/10.1007/s00294-017-0795-3