The Eukaryotic Initiation Factor (eIF) 4G HEAT Domain Promotes Translation Re-initiation in Yeast Both Dependent on and Independent of eIF4A mRNA Helicase

Translation re-initiation provides the molecular basis for translational control of mammalian ATF4 and yeast GCN4 mediated by short upstream open reading (uORFs) in response to eIF2 phosphorylation. eIF4G is the major adaptor subunit of eIF4F that binds the cap-binding subunit eIF4E and the mRNA helicase eIF4A and is also required for re-initiation in mammals. Here we show that the yeast eIF4G2 mutations altering eIF4E- and eIF4A-binding sites increase re-initiation at GCN4 and impair recognition of the start codons of uORF1 or uORF4 located after uORF1. The increase in re-initiation at GCN4 was partially suppressed by increasing the distance between uORF1 and GCN4, suggesting that the mutations decrease the migration rate of the scanning ribosome in the GCN4 leader. Interestingly, eIF4E overexpression suppressed both the phenotypes caused by the mutation altering eIF4E-binding site. Thus, eIF4F is required for accurate AUG selection and re-initiation also in yeast, and the eIF4G interaction with the mRNA-cap appears to promote eIF4F re-acquisition by the re-initiating 40 S subunit. However, eIF4A overexpression suppressed the impaired AUG recognition but not the increase in re-initiation caused by the mutations altering eIF4A-binding site. These results not only provide evidence that mRNA unwinding by eIF4A stimulates start codon recognition, but also suggest that the eIF4A-binding site on eIF4G made of the HEAT domain stimulates the ribosomal scanning independent of eIF4A. Based on the RNA-binding activities identified within the unstructured segments flanking the eIF4G2 HEAT domain, we discuss the role of the HEAT domain in scanning beyond loading eIF4A onto the pre-initiation complex.     

Molecular Cloning and Characterization of a Gene Encoding Eukaryotic Initiation Factor iso4E in Tomato (Solanum lycopersicum)

The eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E, play important roles in protein translation and recently reported to be involved in plant–virus interactions. A cDNA encoding the tomato eIF(iso)4E was cloned based on a tentative consensus (TC170275) in TIGR (), and was designated as SleIF(iso)4E, with an open reading frame of 603 nucleotides encoding a protein of 200 amino acids. The calculated molecular weight of the SleIF(iso)4E protein was 22.85 kD, and the theoretical isoelectric point was 5.76. The amino acid sequence of SleIF(iso)4E showed 66–91% identity with eIF(iso)4Es in pepper, tobacco, pea and maize, and 44–51% identity with eIF4Es from other plants. The phylogenetic relationship and tertiary structure comparisons indicate that SleIF(iso)4E share high homology and strict conserved regions with other members of the eIF4E family, a characteristic of all members of this family. Semi-quantitative RT-PCR showed varying expression levels of SleIF(iso)4E in different tissues. By comparing eIF(iso)4E coding sequences between resistant and susceptible tomato genotypes, correlation between sequence variations and virus resistance was identified. These findings provide good grounds for future research on the role of SleIF(iso)4E in translation initiation and plant–virus interactions. Sequence data of SleIF(iso)4E from this article have been deposited at GenBank under accession number EU119958.     

Deoxyhypusine Modification of Eukaryotic Translation Initiation Factor 5A (eIF5A) Is Essential for Trypanosoma brucei Growth and for Expression of Polyprolyl-containing Proteins

The eukaryotic protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis. Polyamine biosynthesis is essential in T. brucei, and the polyamine spermidine is required for synthesis of a novel cofactor called trypanothione and for deoxyhypusine modification of eukaryotic translation initiation factor 5A (eIF5A). eIF5A promotes translation of proteins containing polyprolyl tracts in mammals and yeast. To evaluate the function of eIF5A in T. brucei, we used RNA interference (RNAi) to knock down eIF5A levels and found that it is essential for T. brucei growth. The RNAi-induced growth defect was complemented by expression of wild-type human eIF5A but not by a Lys-50 mutant that blocks modification by deoxyhypusine. Bioinformatics analysis showed that 15% of the T. brucei proteome contains 3 or more consecutive prolines and that actin-related proteins and cysteine proteases were highly enriched in the group. Steady-state protein levels of representative proteins containing 9 consecutive prolines that are involved in actin assembly (formin and CAP/Srv2p) were significantly reduced by knockdown of eIF5A. Several T. brucei polyprolyl proteins are involved in flagellar assembly. Knockdown of TbeIF5A led to abnormal cell morphologies and detached flagella, suggesting that eIF5A is important for translation of proteins needed for these processes. Potential specialized functions for eIF5A in T. brucei in translation of variable surface glycoproteins were also uncovered. Inhibitors of deoxyhypusination would be expected to cause a pleomorphic effect on multiple cell processes, suggesting that deoxyhypusine/hypusine biosynthesis could be a promising drug target in not just T. brucei but in other eukaryotic pathogens.     

eIF1 Controls Multiple Steps in Start Codon Recognition during Eukaryotic Translation Initiation

Eukaryotic translation initiation factor (eIF) 1 is a central mediator of start codon recognition. Dissociation of eIF1 from the preinitiation complex (PIC) allows release of phosphate from the G-protein factor eIF2, triggering downstream events in initiation. Mutations that weaken binding of eIF1 to the PIC decrease the fidelity of start codon recognition (Sui⁻ phenotype) by allowing increased eIF1 release at non-AUG codons. Consistent with this, overexpression of these mutant proteins suppresses their Sui⁻ phenotypes. Here, we have examined mutations at the penultimate residue of eIF1, G107, that produce Sui⁻ phenotypes without increasing the rate of eIF1 release. We provide evidence that, in addition to its role in gating phosphate release, dissociation of eIF1 triggers conversion from an open, scanning-competent state of the PIC to a stable, closed one. We also show that eIF5 antagonizes binding of eIF1 to the complex and that key interactions of eIF1 with its partners are modulated by the charge at and around G107. Our data indicate that eIF1 plays multiple roles in start codon recognition and suggest that prior to AUG recognition it prevents eIF5 from binding to a key site in the PIC required for triggering downstream events.     

General RNA‐binding proteins have a function in poly(A)‐binding protein‐dependent translation

The interaction between the poly(A)‐binding protein (PABP) and eukaryotic translational initiation factor 4G (eIF4G), which brings about circularization of the mRNA, stimulates translation. General RNA‐binding proteins affect translation, but their role in mRNA circularization has not been studied before. Here, we demonstrate that the major mRNA ribonucleoprotein YB‐1 has a pivotal function in the regulation of eIF4F activity by PABP. In cell extracts, the addition of YB‐1 exacerbated the inhibition of 80S ribosome initiation complex formation by PABP depletion. Rabbit reticulocyte lysate in which PABP weakly stimulates translation is rendered PABP‐dependent after the addition of YB‐1. In this system, eIF4E binding to the cap structure is inhibited by YB‐1 and stimulated by a nonspecific RNA. Significantly, adding PABP back to the depleted lysate stimulated eIF4E binding to the cap structure more potently if this binding had been downregulated by YB‐1. Conversely, adding nonspecific RNA abrogated PABP stimulation of eIF4E binding. These data strongly suggest that competition between YB‐1 and eIF4G for mRNA binding is required for efficient stimulation of eIF4F activity by PABP.     

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: A reappraisal of translation initiation during mitosis

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G₁/S boundary) or the Cdk1 inhibitor, RO3306 (G₂/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.