eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

Abstract

Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

Keywords: 14-3-3 protein; kinetoplastid; mRNA cap; social motility; translation initiation factor.

FIGURE 1.

TbEIF4E5 and TbEIF4E6 are shorter than the eIF4E homologs from yeast and human. (A) Alignment of the sequences was performed using Clustal W. 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. Dashes represent spaces that were inserted to allow better alignment. Asterisks represent tryptophan residues conserved in the eIF4E protein family. Arrowheads indicate nontryptophan residues required for the interaction with the cap structure: E103 hydrogen bonded with guanine; and, basic residues at positions 112, 157, and 162 with the phosphate bridge (Marcotrigiano et al. 1997). Thin arrows indicate conserved nontryptophan residues shown to be involved in eIF4G binding (Marcotrigiano et al. 1999). GenBank Accession numbers: Hs (human) eIF4E-1, NP_001959; Sc (yeast) eIF4E, NP_014502. (B) In vitro cap-binding ability of recombinant TbE5. The fluorescence-titration curves with four cap analogs were determined by fluorescence-binding assays. The protein fluorescence was excited at 280 nm and observed at 340 nm. The WT mRNA cap in trypanosomes was represented by hypermethylated cap 4, while the typical eukaryotic cap structure was represented by both the m⁷GTP and m⁷GpppA cap 0 structures. The nonmethylated GTP served as a negative control for cap 0-specific binding.

FIGURE 2.

Direct interaction of TbEIF4E5 with two of the five T. brucei EIF4G homologs. Interactions between TbE5 and the five TbEIF4G homologs were challenged 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. Interaction strength is inferred by colony size: ≥2 mm = strong; 1–2 mm = moderate; ≤1 mm = weak.

FIGURE 3.

TbEIF4E5 is present in high molecular weight complexes. (A) Blue Native gel electrophoresis of cell extracts from transfected T. brucei containing PTP-tagged proteins. Lysates were run through Blue Native gels, transferred to nitrocellulose membranes and probed with antibody directed against the protein A domain of the PTP tag (TbE5, TbG1, TbG2 = E5, G1, G2, respectively). The migration of size standards is indicated in kDa. (B) The components that co-purified with TbEIF4E5 and TbEIF4G2. The RNA (black line; black circle represents 5′ end cap 4) is recognized by the cap-binding eIF4E component TbE5 (dark gray circle, E5). The eIF4G-like scaffold protein TbG2 (light gray rectangle, G2) interacts directly with TbE5, as indicated by overlap of the respective shapes. Other TbE5-associated proteins are shown as ovals. Overlap of the ovals with other shapes is speculative and based on the assumed scaffold function of TbG2, and shapes do not reflect protein size. The placement of Tb17.9 on the RNA is speculative, as indicated by the “?”. (C) The TbEIF4E5/TbEIF4G1 dynamic complex model. The 117.5-kDa protein in association with the TbG1 scaffold may be regulated by the presence of the 14-3-3 II homodimer, the binding of which is dependent on TbG1 phosphorylation. The dotted arrows highlight the either/or aspect of the complexes. (D) Yeast two-hybrid analysis of Tb117.5 with TbE5 or TbG1 in both bait and prey orientations. Controls and interpretations are shown in Figure 2.

FIGURE 4.

TbEIF4E5 is cytosolic in T. brucei procyclic cells. Subcellular localization was determined by indirect immunofluorescence using antibody against the protein A component of the PTP tag on the TbE5 fusion protein. Nontransfected control YTAT cells served as the negative control. Nuclear and kinetoplast DNA was visualized by counterstaining with DAPI.

FIGURE 5.

TbEIF4E5 does not have a primary role in translation. (A) Growth of TbE5 RNAi tetracycline (Tet) induced and uninduced cells measured over 7 d. (B) Assessment of protein knockdown by RNAi using the TbE5^PTP/+ background cell line. SDS-PAGE analysis of protein levels at days 2, 3, and 4 post-induction. TbE5-PTP was detected by anti-Protein A antibody (α-ProtA). Serial twofold dilutions of uninduced samples at day 2 are shown for comparison. Levels of TbEIF4A1 are included as protein loading controls. (C) Metabolic labeling of cultures induced for TbE5 RNAi knockdown as measured by ³⁵S-methionine incorporation at 4 and 7 d post-induction.

FIGURE 6.

Reduction of TbEIF4E5 results in motility defects. (A) Culture turbidity was measured to assess cell settling in nonshaken cultures. Cells induced for RNAi against TbE5 (white column) were compared with noninduced culture (gray column) and WT cells (black column). Standard error was determined for experiments performed in triplicate. (B) Representative soft agarose plate Social Motility assays for TbE5 RNAi-uninduced (−Tet) or RNAi-induced cells (+Tet) 5 d post-plating. The − plate was scored as showing 10 radial projections, a typical manifestation of social motility in T. brucei. (C) Graphical summary of two sets of SoMo assays indicating means and standard errors. Symbols represent the number of radial projections from the site of inoculation: Open squares represent uninduced (−Tet); the single filled triangle represents all 14 induced samples (+Tet).

FIGURE 7.

Identified domains in two hypothetical TbEIF4E5/TbEIF4G1 complex members. Schematic locations of the conserved structural domains in the TbE5-associated 47.5-kDa and 117.5-kDa proteins as predicted by Phyre² analysis, along with two proteins sharing domains with the 117.5-kDa protein. FD, ferredoxin-like fold RNA-binding domain; yth, yth domain RNA-binding motif; GTase, guanylyltransferase; Z, zinc finger; MTase, m⁷G methyltransferase; NTPase, nucleoside triphosphate hydrolase.