The eIF3 complex of Trypanosoma brucei : composition conservation does not imply the conservation of structural assembly and subunits function.

The multisubunit eukaryotic initiation factor 3 (eIF3) plays multiple roles in translation but is poorly understood in trypanosomes. The putative subunits eIF3a and eIF3f of Trypanosoma brucei ( Tb IF3a and Tb IF3f) were overexpressed and purified, and 11 subunits were identified, Tb IF3a through l minus j, which form a tight complex. Both Tb IF3a and Tb IF3f are essential for the viability of T. brucei RNAi knockdown of either of them severely reduced total translation and the ratio of the polysome/80S peak area. Tb IF3f and Tb IF3a RNAi cell lines were modified to express tagged- Tb IF3a and - Tb IF3f, respectively. RNAi in combination with affinity purification assays indicated that both subunits are variably required for Tb IF3 stability and integrity. The relative abundance of other subunits in the Tb IF3f-tag complex changed little upon Tb IF3a depletion; while only subunits Tb IF3b, i, and e copurified comparably with Tb IF3a-tag upon Tb IF3f depletion. A genome-wide UV-crosslinking assay showed that several Tb IF3 subunits have direct RNA-binding activity, with Tb IF3c showing the strongest signal. In addition, CrPV IRES, but neither EMCV IRES nor HCV IRES, was found to mediate translation in T. brucei These results together imply that the structure of Tb IF3 and the subunits function have trypanosome-specific features, although the composition is evolutionarily conserved.