Two related trypanosomatid eIF4G homologues have functional differences compatible with distinct roles during translation initiation

Abstract

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.

Characterization of RBP9 and RBP10, two developmentally regulated RNA-binding proteins in Trypanosoma brucei

“…Streptavidin-POD (Jackson Immunoresearch) was diluted 1/1000, anti-myc 1/1000, anti-BiP 1/1 0000, anti-P0 1/2500 and anti-eIF4A1 used as loading control 1/5000 [48]. …”

The suppressive cap-binding-complex factor 4EIP is required for normal differentiation

“…EIF4E3 and EIF4E4 are present in roughly equimolar ratios, or a slight excess, relative to mRNA (55,56). RNAi experiments showed that EIF4E3 is essential in both forms whereas for EIF4E4, a growth defect was seen only in bloodstream forms (57). The T. brucei EIF4E5-EIF4G2, EIF4E5-EIF4G1 (58), and EIF4E6-EIF4G5 (59) complexes might be implicated in translation of specific mRNA subsets, but these have yet to be characterised; alternative functions are also conceivable.…”

“…Since there are 3 times fewer EIF4E1 molecules per cell than there are mRNAs (55,66), it cannot be a major general translation initiation factor. Depletion of T. brucei eIF4E1 by RNAi halted growth of bloodstream forms and slowed growth of procyclic forms (57). EIF4E1 and 4EIP (Tb927.9.11050) were found to be extremely strong repressors when tethered to reporter mRNAs (67,68).…”

Phosphorylation and interactions associated with the control of theLeishmaniaPoly-A Binding Protein 1 (PABP1) function during translation initiation

“…The six conserved eIF4Es (EIF4E1 through EIF4E6) and five conserved eIF4Gs (EIF4G1 through EIF4G5) differ substantially in sequence and in binding partners [42][43][44][45][46]. Two distinct eIF4Flike complexes, centered on the interactions between EIF4E4/ EIF4G3 and EIF4E3/EIF4G4, have been characterized with properties which implicate them during translation initiation [47][48][49][50][51]. In both Leishmania and Trypanosoma species, the EIF4E4/EIF4G3 complex has been shown to be associated with PABP1 [35,36,49] and the Leishmania PABP1 has been seen to bind directly to EIF4E4, through an interaction unknown from other eukaryotes [49].…”