The four trypanosomatid eIF4E homologues fall into two separate groups, with distinct features in primary sequence and biological properties

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.

Graphical abstractThe four Trypanosoma brucei eIF4E homologues differ in subcellular localization, impact on translation and viability after RNAi and in ability to form eIF4F complexes.Download high-res image (194KB)Download full-size imageResearch highlights▶ Sequence analysis and subcellular localization separate the four eIF4Es in two groups. ▶ RNAi data in procyclic cells highlight a major role for TbEIF4E3 in parasite survival. ▶ TbEIF4E1, 3 and 4 are essential for survival and proliferation of bloodstream forms. ▶ Metabolic labeling implicates TbEIF4E3 and 4 in protein synthesis. ▶ TbEIF4E3 and 4 interact with eIF4G homologues forming multiple eIF4F complexes.