[Abstract unavailable

The cloning is described of two related human complementary DNAs encoding polypeptides that interact specifically with the translation initiation factor eIF-4E, which binds to the messenger RNA 5'-cap structure. Interaction of these proteins with eIF-4E inhibits translation but treatment of cells with insulin causes one of them to become hyperphosphorylated and dissociate from eIF-4E, thereby relieving the translational inhibition. The action of this new regulator of protein synthesis is therefore modulated by insulin, which acts to stimulate the overall rate of translation and promote cell growth.

mRNA translation is thought to be the most energy-consuming process in the cell. Translation and energy metabolism are dysregulated in a variety of diseases including cancer, diabetes, and heart disease. However, the mechanisms that coordinate translation and energy metabolism in mammals remain largely unknown. The mechanistic/mammalian target of rapamycin complex 1 (mTORC1) stimulates mRNA translation and other anabolic processes. We demonstrate that mTORC1 controls mitochondrial activity and biogenesis by selectively promoting translation of nucleus-encoded mitochondria-related mRNAs via inhibition of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BPs). Stimulating the translation of nucleus-encoded mitochondria-related mRNAs engenders an increase in ATP production capacity, a required energy source for translation. These findings establish a feed-forward loop that links mRNA translation to oxidative phosphorylation, thereby providing a key mechanism linking aberrant mTOR signaling to conditions of abnormal cellular energy metabolism such as neoplasia and insulin resistance.

Eukaryotic translation initiation factor 4E (eIF-4E), which possesses cap-binding activity, functions in the recruitment of mRNA to polysomes as part of a three-subunit complex, eIF-4F (cap-binding complex). eIF-4E is the least abundant of all translation initiation factors and a target of growth regulatory pathways. Recently, two human cDNAs encoding novel eIF-4E-binding proteins (4E-BPs) which function as repressors of capdependent translation have been cloned. Their interaction with eIF-4E is negatively regulated by phosphorylation in response to cell treatment with insulin or growth factors. The present study aimed to characterize the molecular interactions between eIF-4E and the other subunits of eIF-4F and to similarly characterize the molecular interactions between eIF-4E and the 4E-BPs. A 49-amino-acid region of eIF-4␥, located in the N-terminal side of the site of cleavage by Picornaviridae protease 2A, was found to be sufficient for interacting with eIF-4E. Analysis of deletion mutants in this region led to the identification of a 12-amino-acid sequence conserved between mammals and Saccharomyces cerevisiae that is critical for the interaction with eIF-4E. A similar motif is found in the amino acid sequence of the 4E-BPs, and point mutations in this motif abolish the interaction with eIF-4E. These results shed light on the mechanisms of eIF-4F assembly and on the translational regulation by insulin and growth factors.A ubiquitous feature of all eukaryotic cellular mRNAs is the presence at their 5Ј end of a cap structure, m7GpppX (where X is any nucleotide). The cap facilitates mRNA binding to ribosomes, a step which is rate limiting in translation (for reviews, see references 18 and 20). The cap is bound by the eukaryotic initiation factor 4F (eIF-4F) protein complex, which consists in mammals of three polypeptides: eIF-4E (a 25-kDa cap-binding protein), eIF-4␥ (also known as p220 in mammals), and eIF-4A (a 46-kDa RNA helicase). eIF-4F, in conjunction with eIF-4B, exhibits RNA helicase activity. Evidence suggests that this activity mediates mRNA unwinding in the vicinity of the cap structure in an ATP-dependent manner and facilitates binding of the 40S ribosomal subunit (26, 33). The 40S subunit is then thought to scan the mRNA in the 5Ј-to-3Ј direction until it encounters the initiator AUG codon. This series of steps constitutes the cap-dependent pathway of translation initiation (26,33).eIF-4E is the least abundant of all translation initiation factors, and its overexpression leads to deregulation of cell growth in HeLa cells (6) and to transformation of NIH 3T3 cells and CHO cells (5, 23) or rat primary fibroblasts, the latter in combination with v-myc or E1A (24). An attractive model for eIF-4E transforming activity is that overexpression of eIF-4E results in increased translation of mRNAs which are normally repressed, such as protooncogene mRNAs. eIF-4E activity is upregulated upon treatment of cells with growth factors, mitogens, and phorbol esters, which correlates with an increase in translatio...

eIF‐4A is a translation initiation factor that exhibits bidirectional RNA unwinding activity in vitro in the presence of another translation initiation factor, eIF‐4B and ATP. This activity is thought to be responsible for the melting of secondary structure in the 5′ untranslated region of eukaryotic mRNAs to facilitate ribosome binding. eIF‐4A is a member of a fast growing family of proteins termed the DEAD family. These proteins are believed to be RNA helicases, based on the demonstrated in vitro RNA helicase activity of two members (eIF‐4A and p68) and their homology in eight amino acid regions. Several related biochemical activities were attributed to eIF‐4A: (i) ATP binding, (ii) RNA‐dependent ATPase and (iii) RNA helicase. To determine the contribution of the highly conserved regions to these activities, we performed site‐directed mutagenesis. First we show that recombinant eIF‐4A, together with recombinant eIF‐4B, exhibit RNA helicase activity in vitro. Mutations in the ATPase A motif (AXXXXGKT) affect ATP binding, whereas mutations in the predicted ATPase B motif (DEAD) affect ATP hydrolysis. We report here that the DEAD region couples the ATPase with the RNA helicase activity. Furthermore, two other regions, whose functions were unknown, have also been characterized. We report that the first residue in the HRIGRXXR region is involved in ATP hydrolysis and that the SAT region is essential for RNA unwinding. Our results suggest that the highly conserved regions in the DEAD box family are critical for RNA helicase activity.

PHAS-I is a heat-stable protein (relative molecular mass approximately 12,400) found in many tissues. It is rapidly phosphorylated in rat adipocytes incubated with insulin or growth factors. Nonphosphorylated PHAS-I bound to initiation factor 4E (eIF-4E) and inhibited protein synthesis. Serine-64 in PHAS-I was rapidly phosphorylated by mitogen-activated (MAP) kinase, the major insulin-stimulated PHAS-I kinase in adipocyte extracts. Results obtained with antibodies, immobilized PHAS-I, and a messenger RNA cap affinity resin indicated that PHAS-I did not bind eIF-4E when serine-64 was phosphorylated. Thus, PHAS-I may be a key mediator of the stimulation of protein synthesis by the diverse group of agents and stimuli that activate MAP kinase.

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SUMMARYProtein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis consists of the eukaryotic translation initiation factor 4E (eIF4E) binding to the 7-methylguanosine (m7-GpppG) 5′cap of mRNAs1,2. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms3–6. While the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells7,8. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition9. Here, we uncover an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. Hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. PAR-CLIP10 analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array mRNAs, including the epidermal growth factor receptor (EGFR). Once assembled at the rHRE, HIF-2α/RBM4/eIF4E2 captures the 5′cap and targets mRNAs to polysomes for active translation thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program whereby oxygen tension switches the basic translation initiation machinery.