Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog

Abstract

T he operon arrangement used by prokaryotes is an elegant solution to the question of regulated gene expression, with coordinated transcription of genes encoding enzymes within a given metabolic pathway under the control of a single promoter. In contrast, the majority of eukaryotes evolved independent promoters to control the expression of individual genes, and promoter types fall into classes that are activated or repressed in synchrony with functionally linked genes. Kinetoplastids employ an unusual blend of these two strategies, the constitutive transcription of polycistronic gene clusters that, apart from tandem gene arrays, typically show no discernible biochemical linkage within arrays (1, 2). The result is the virtual absence of genetic control at the level of gene transcription for mRNAs transcribed by RNA polymerase II (3, 4). Trypanosoma brucei has circumvented this limitation for the expression of a set of virulence factors associated with the variant surface glycoproteins. This family provides the coat on the cell surface and cycles a single member over time to allow this parasite to evade the host immune system. RNA polymerase I promoters provide temporal control to this gene set (5, 6). This unusual choice of polymerase is available to trypanosomes because of the mechanism that also provides a complex mRNA cap structure to all nuclear transcripts, namely, trans splicing of the spliced leader (SL) RNA (7).The SL RNA is a small, independently transcribed molecule that is the source of the hypermethylated cap 4 structure that defines nucleus-derived mRNA in kinetoplastids (8). The cap 4 structure consists of cap 0 followed by 2=-O-methylation of the first four transcribed nucleotides and an additional three methylations on the first and fourth bases (9). The first 39 nucleotides are transferred by trans splicing to each gene transcript in a polycistronic array, which, coupled with 3= polyadenylation (10), results in a monocistronic mRNA population looking very much like that from any other eukaryote with a few extra 5= methylations. Other eukaryotes widely separated from each other in evolutionary terms use this combination of polycistronic transcription and trans splicing of their own flavor of SL (11-13).RNA cap formation requires a minimum of three enzymatic activities, a triphosphatase to remove the gamma phosphate of the primary transcript, a guanylyltransferase to attach an inverted GTP cap via a triphosphate bridge, and a methyltransferase to complete the m 7 G modification that defines cap 0 (14). This trio of activities is found in various combinations in different systems, including three separate proteins in yeast, a pairing of the first and second activities in metazoa and plants or the second and third

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

“…This conservation is in striking contrast with the divergent N and C-terminal regions of the other 3 trypanosomatid eIF4G homologues (EIF4G1, EIF4G2 and EIF4G5) which have recently been seen to participate in novel eIF4F-like complexes that do not seem to have primary roles in translation. 40,41 The presence of putative MA3/ HEAT-2 and W2/HEAT-3 domains in at least 2 trypanosomatid eIF4G homologues confirms the likely ancient origin of the eIF4G tripartite structure, despite the fact that both MA3 and W2 are missing from yeast eIF4G and the W2 domain is not found in plant eIF4G homologues. 32 The evidence presented clearly distinguishes EIF4G3 and EIF4G4 functionally.…”

“…More recently EIF4G1, EIF4G2 and EIF4G5 have been shown to form complexes with 2 novel eIF4E homologues, EIF4E5 and EIF4E6, but they have not been linked to the translation initiation process. 40,41 Here, the functional properties and individual roles of EIF4G3 and EIF4G4 have been further investigated in both L. major and T. brucei. First, binding to eIF4Es, eIF4A and PABPs were investigated in vitro.…”

Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA‐binding proteome

“…Interestingly, several eIF4 initiation factors tethered to the 3'UTR can also increase gene expression. The trypanosome genome encodes multiple homologues for the eIF4A (two), eIF4E (six) and eIF4G (five) subunits (Dhalia et al ., ; Freire et al ., ; Moura et al ., ). However, only eIF4E3 and eIF4E4, which are in complex with eIF4G4 and eIF4G3, respectively, are thought to have a main role in the translation initiation complex (Zinoviev and Shapira, ).…”

Structural basis for LeishIF4E-1 modulation by an interacting protein in the human parasite Leishmania major

“…Trypanosomatids encode several isoforms of the same initiation factor; there are six highly diverged paralogs of the human eIF4E cap-binding protein (LeishIF4E-1 to LeishIF4E-6). These have a small degree of conservation among themselves (40–60%) and also differ significantly from their counterparts in higher eukaryotes (30–40% sequence identity) (Supplementary Figure S1A) (24,26,27). Of the six paralogs, LeishIF4E-1 is highly expressed in axenic amastigotes and binds the m 7 GTP at 37°C, suggesting its involvement in translation initiation in amastigotes (23).…”