eIF4E, the mRNA 5 cap-binding protein, is regulated by its binding protein (4E-BP), a downstream target of phosphatidylinositol-3-OH kinase [PI(3)K] signaling. We show thatDrosophila 4E-BP (d4E-BP) activity becomes critical for survival under dietary restriction and oxidative stress, and is linked to life span. The Drosophila forkhead transcription factor (dFOXO) activates d4E-BP transcription. We show that ectopic expression of d4E-BP in dFOXO-null flies restores oxidative stress resistance to control levels. Thus, d4E-BP is an important downstream effector of a dFOXO phenotype, and regulation of translation by eIF4E is vital during environmental stress. A rapid response is a crucial early line of defense in preventing cellular death in situations of stress . Translational regulation allows an organism to generate quick responses to environmental cues by controlling the expression of protein from existing cellular mRNAs (Johnstone and Lasko 2001). Translation initiation of most eukaryotic mRNAs requires binding of eIF4F, a protein complex made up of eIF4A, eIF4G, and eIF4E, to the 5Ј cap structure (Gingras et al. 1999). eIF4E activity is highly regulated both by Mnk1/Mnk2-dependent phosphorylation (Waskiewicz et al. 1997) and by repressor proteins termed eIF4E-binding proteins (4E-BPs), which compete with eIF4G for the same binding site on eIF4E (Haghighat et al. 1995;Mader and Sonenberg 1995). 4E-BPs themselves are negatively regulated by phosphorylation, and are downstream effectors of the PI3K/TOR pathway (Miron et al. 2001). Under nutritionally favorable conditions, the evolutionarily conserved TOR pathway is active and results in 4E-BP phosphorylation. This prevents 4E-BP binding to eIF4E, thus upregulating translation (Schmelzle and Hall 2000;Hay and Sonenberg 2004). Conversely, poor nutrition causes inhibition of the TOR pathway, such that unphosphorylated 4E-BP represses translation through eIF4E binding.Activation of the PI3K pathway stimulates Akt activity. Akt directly phosphorylates the forkhead transcription factor dFOXO, which is a transcriptional activator of d4E-BP in flies, resulting in reduced d4E-BP transcription (Jünger et al. 2003;Puig et al. 2003). Akt also activates TOR through tuberous sclerosis complex 2 (TSC2), which functions as a GTPase-activating protein (GAP) for the GTPase protein Rheb that activates TOR (Hay and Sonenberg 2004). Thus, activation of the PI3K pathway in Drosophila represses both the expression of the d4E-BP gene and the activity of d4E-BP protein.Here we investigated whether d4E-BP is essential under starvation and oxidative stress conditions, because dFOXO activates the transcription of d4E-BP and d4E-BP mRNA levels increase upon starvation (Zinke et al. 2002). We also provide evidence that d4E-BP activity is linked to life span, as overexpression of dFOXO is linked to increased longevity (Giannakou et al. 2004;Hwangbo et al. 2004). We present data that support both connections and indicate that d4E-BP is the critical effector of the dFOXO-induced stress-sensitive...

Translational control plays an important role in cell growth and tumorigenesis. Cap-dependent translation initiation of mammalian mRNAs with structured 5 UTRs requires the DExH-box protein, DHX29, in vitro. Here we show that DHX29 is important for translation in vivo. Down-regulation of DHX29 leads to impaired translation, resulting in disassembly of polysomes and accumulation of mRNA-free 80S monomers. DHX29 depletion also impedes cancer cell growth in culture and in xenografts. Thus, DHX29 is a bona fide translation initiation factor that potentially can be exploited as a target to inhibit cancer cell growth.I nitiation is a tightly regulated rate-limiting step in the translation of eukaryotic mRNAs. Ribosome recruitment to the mRNA commences with binding of translation initiation factor 4F (eIF4F) to the 7-methyl guanosine cap structure, which is present at the 5Ј end of all nuclear-encoded eukaryotic mRNAs (1). eIF4F (comprising the cap-binding protein eIF4E, the DEAD-box RNA helicase eIF4A and eIF4G, a scaffold for binding eIF4E and eIF4A) binds to the cap, unwinds (with the aid of eIF4A) the cap-proximal region of the mRNA, and, through interaction with the ribosome-bound eIF3, recruits the 40S ribosomal subunit to the mRNA (2-4). The 40S subunit then scans the 5Ј UTR in a 5Ј to 3Ј direction until it encounters an initiation codon. A subsequent joining of the 60S ribosomal subunit and release of eIFs result in formation of an elongationcompetent 80S ribosome.Secondary structures in 5ЈUTRs of mRNAs are thought to become unwound to allow ribosomal complexes to move along the mRNA in search of the initiation codon. Thus, in addition to its role in the initial attachment of ribosomal complexes to mRNA, eIF4A is believed to assist ribosomal complexes during scanning (5). Recent observations suggest that the process of eukaryotic initiation requires additional members of the DEAD/DExH-box protein family; for instance, a DEAD-box protein, yeast Ded1, and its mammalian homologue, DDX3, are biochemically and genetically implicated in translation initiation on long structured 5ЈUTRs (6), and another DExH-box protein, DHX29, strongly stimulates cap-dependent initiation on mRNAs with structured 5ЈUTRs in vitro (7). Here we studied the importance of DHX29 for translation in vivo and characterized it as a novel factor required for cell proliferation. Results DHX29 Is a Ubiquitously Expressed Cytoplasmic Protein That Associ-ates with the 40S Ribosomal Subunit. DHX29 has been found to interact with the 40S ribosomal subunit in vitro and to associate with 40S ribosomal complexes in a rabbit reticulocyte lysate (7). To determine how general these findings are, we examined the distribution of DHX29 in polysome preparations from HeLa cells using 2 commercial DHX29 antibodies (Fig. 1A). The specificity of these antibodies to human DHX29 was confirmed by immunoblotting against the purified native protein [supporting information (SI) Fig. S1] and recombinant DHX29, as well as by the loss of immunoreactivity following DHX29 RNAi ...

Translation is a fundamental step in gene expression, and translational control is exerted in many developmental processes. Most eukaryotic mRNAs are translated by a cap-dependent mechanism, which requires recognition of the 5=-cap structure of the mRNA by eukaryotic translation initiation factor 4E (eIF4E). eIF4E activity is controlled by eIF4E-binding proteins (4E-BPs), which by competing with eIF4G for eIF4E binding act as translational repressors. Here, we report the discovery of Mextli (Mxt), a novel Drosophila melanogaster 4E-BP that in sharp contrast to other 4E-BPs, has a modular structure, binds RNA, eIF3, and several eIF4Es, and promotes translation. Mxt is expressed at high levels in ovarian germ line stem cells (GSCs) and early-stage cystocytes, as is eIF4E-1, and we demonstrate the two proteins interact in these cells. Phenotypic analysis of mxt mutants indicates a role for Mxt in germ line stem cell (GSC) maintenance and in early embryogenesis. Our results support the idea that Mxt, like eIF4G, coordinates the assembly of translation initiation complexes, rendering Mxt the first example of evolutionary convergence of eIF4G function.

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Insects are part of the earliest faunas that invaded terrestrial environments and are the first organisms that evolved controlled flight. Nowadays, insects are the most diverse animal group on the planet and comprise the majority of extant animal species described. Moreover, they have a huge impact in the biosphere as well as in all aspects of human life and economy; therefore understanding all aspects of insect biology is of great importance. In insects, as in all cells, translation is a fundamental process for gene expression. However, translation in insects has been mostly studied only in the model organism Drosophila melanogaster. We used all publicly available genomic sequences to investigate in insects the distribution of the genes encoding the cap-binding protein eIF4E, a protein that plays a crucial role in eukaryotic translation. We found that there is a diversity of multiple ortholog genes encoding eIF4E isoforms within the genus Drosophila. In striking contrast, insects outside this genus contain only a single eIF4E gene, related to D. melanogaster eIF4E-1. We also found that all insect species here analyzed contain only one Class II gene, termed 4E-HP. We discuss the possible evolutionary causes originating the multiplicity of eIF4E genes within the genus Drosophila.