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