The two eIF4A helicases in Trypanosoma brucei are functionally distinct

Abstract

Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5′ capped mini-exon which is linked to the 3′ cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits the small ribosomal subunit. TbEIF4AI is a very abundant predominantly cytoplasmic protein (over 1 × 105 molecules/cell) and depletion to ∼10% of normal levels through RNA interference dramatically reduces protein synthesis one cell cycle following double-stranded RNA induction and stops cell proliferation. In contrast, TbEIF4AIII is a nuclear, moderately expressed protein (∼1–2 × 104 molecules/cell), and its depletion stops cellular proliferation after approximately four cell cycles. Ectopic expression of a dominant negative mutant of TbEIF4AI, but not of TbEIF4AIII, induced a slow growth phenotype in transfected cells. Overall, our results suggest that only TbEIF4AI is involved in protein synthesis while the properties and sequence of TbEIF4AIII indicate that it may be the orthologue of eIF4AIII, a component of the exon junction complex in mammalian cells.

Dynamic Behavior ofArabidopsiseIF4A-III, Putative Core Protein of Exon Junction Complex: Fast Relocation to Nucleolus and Splicing Speckles under Hypoxia

“…Colocalization of Arabidopsis Y14 and Mago proteins with a marker for nuclear speckles SRp34 has been previously demonstrated (Park and Muench, 2007), and nucleolar localization was confirmed by both proteomic studies and transient expression of GFP fusions (Pendle et al, 2005). The nuclear localization is a unique feature of eIF4A-III, since RNA helicase with high sequence similarity, eIF4AI, is predominantly cytoplasmic (Dhalia et al, 2006) like some other proteins from the same family (Koroleva et al, 2005). Thus, various lines of evidence confirm that At3g19760 is the Arabidopsis eIF4A-III ortholog, and we refer to it subsequently as eIF4A-III.…”

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

“…As with the TbEIF4E proteins, in kinetoplastids the TbEIF4G family is expanded relative to other eukaryotes, containing five identified members (Dhalia et al 2006). The direct interaction between the TbE5 and the TbG1 and TbG2 homologs predicted by the initial yeast two-hybrid assay was confirmed in vivo.…”

“…Recombinant TbE5 was expressed from the p2171 plasmid (Dhalia et al 2006). For interaction assays, the TbE5 and TbEIF4G homolog ORFs were amplified by PCR, and cloned into the yeast two-hybrid vectors pGAD and pGBK.…”

Functional Characterization of Three Leishmania Poly(A) Binding Protein Homologues with Distinct Binding Properties to RNA and Protein Partners

“…9,2010 CHARACTERIZATION OF THREE LEISHMANIA PABP HOMOLOGUES 1485 10% fetal calf serum. Procyclic forms of T. brucei Lister 427(pLEW29, pLEW13) (62) were maintained as previously described (19). Fluorescence microscopy.…”