Translation initiation in eukaryotes requires eIF4E, the cap binding protein, which mediates its function through an interaction with the scaffolding protein eIF4G, as part of the eIF4F complex. In trypanosomatids, four eIF4E homologues have been described but the specific function of each is not well characterized. Here, we report a study of these proteins in Trypanosoma brucei (TbEIF4E1 through 4). At the sequence level, they can be assigned to two groups: TbEIF4E1 and 2, similar in size to metazoan eIF4E1; and TbEIF4E3 and 4, with long N-terminal extensions. All are constitutively expressed, but whilst TbEIF4E1 and 2 localize to both the nucleus and cytoplasm, TbEIF4E3 and 4 are strictly cytoplasmic and are also more abundant. After knockdown through RNAi, TbEIF4E3 was the only homologue confirmed to be essential for viability of the insect procyclic form. In contrast, TbEIF4E1, 3 and 4 were all essential for the mammalian bloodstream form. Simultaneous RNAi knockdown of TbEIF4E1 and 2 caused cessation of growth and death in procyclics, but with a delayed impact on translation, whilst knockdown of TbEIF4E3 alone or a combined TbEIF4E1 and 4 knockdown led to substantial translation inhibition which preceded cellular death by several days, at least. Only TbEIF4E3 and 4 were found to interact with T. brucei eIF4G homologues; TbEIF4E3 bound both TbEIF4G3 and 4 whilst TbEIF4E4 bound only to TbEIF4G3. These results are consistent with TbEIF4E3 and 4 having distinct but relevant roles in initiation of protein synthesis.

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The poly(A)-blnding pin (PABP), in a complx with the 3' poly(A) tall of eUkaryotic mRNAs, plays important roles in the contol of translation and message stability. All

Trypanosomatid protozoans are reliant on posttranscriptional processes to control gene expression. Regulation occurs at the levels of mRNA processing, stability, and translation, events that may require the participation of the poly(A) binding protein (PABP). Here, we have undertaken a functional study of the three distinct Leishmania major PABP (LmPABP) homologues: the previously described LmPABP1; LmPABP2, orthologous to the PABP described from Trypanosoma species; and LmPABP3, unique to Leishmania. Sequence identity between the three PABPs is no greater than 40%. In assays measuring binding to A-rich sequences, LmPABP1 binding was poly(A) sensitive but heparin insensitive; LmPABP2 binding was heparin sensitive and less sensitive to poly(A), compatible with unique substitutions observed in residues implicated in poly(A) binding; and LmPABP3 displayed intermediate properties. All three homologues are simultaneously expressed as abundant cytoplasmic proteins in L. major promastigotes, but only LmPABP1 is present as multiple isoforms. Upon transcription inhibition, LmPABP2 and -3 migrated to the nucleus, while LmPABP1 remained predominantly cytoplasmic. Immunoprecipitation assays showed an association between LmPABP2 and -3. Although the three proteins bound to a Leishmania homologue of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) (LmEIF4G3) in vitro, LmPABP1 was the only one to copurify with native LmEIF4G3 from cytoplasmic extracts. Functionality was tested using RNA interference (RNAi) in Trypanosoma brucei, where both orthologues to LmPABP1 and -2 are required for cellular viability. Our results indicate that these homologues have evolved divergent functions, some of which may be unique to the trypanosomatids, and reinforces a role for LmPABP1 in translation through its interaction with the eIF4G homologue.

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.

Abbreviations: eIF, eukaryotic Initiation Factor, PABP, Poly(A) Binding Protein, MIF4G, Middle domain of eukaryotic Initiation Factor 4G, HEAT domain, Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A) and the yeast kinase TOR1, GST, Glutathione-S-TransferaseIn higher eukaryotes, eIF4A, eIF4E and eIF4G homologues interact to enable mRNA recruitment to the ribosome. eIF4G acts as a scaffold for these interactions and also interacts with other proteins of the translational machinery. Trypanosomatid protozoa have multiple homologues of eIF4E and eIF4G and the precise function of each remains unclear. Here, 2 previously described eIF4G homologues, EIF4G3 and EIF4G4, were further investigated. In vitro, both homologues bound EIF4AI, but with different interaction properties. Binding to distinct eIF4Es was also confirmed; EIF4G3 bound EIF4E4 while EIF4G4 bound EIF4E3, both these interactions required similar binding motifs. EIF4G3, but not EIF4G4, interacted with PABP1, a poly-A binding protein homolog. Work in vivo with Trypanosoma brucei showed that both EIF4G3 and EIF4G4 are cytoplasmic and essential for viability. Depletion of EIF4G3 caused a rapid reduction in total translation while EIF4G4 depletion led to changes in morphology but no substantial inhibition of translation. Sitedirected mutagenesis was used to disrupt interactions of the eIF4Gs with either eIF4E or eIF4A, causing different levels of growth inhibition. Overall the results show that only EIF4G3, with its cap binding partner EIF4E4, plays a major role in translational initiation.

The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the ≈ 60‐kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino‐acid sequence confirmed its identity as an α‐glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19‐nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full‐length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N‐terminal 45‐kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense‐mediated decay pathway of mRNA degradation. In‐gel assays confirmed that a single α‐glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N‐terminus of the receptor.

Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5′ capped mini-exon which is linked to the 3′ cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 × 105 molecules/cell) and depletion to ∼10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (∼1–2 × 104 molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.