The eIF3 complex of <i>Trypanosoma brucei</i>: composition conservation does not imply the conservation of structural assembly and subunits function

Abstract

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.