Two structurally atypical HEAT domains in the C-terminal portion of human eIF4G support binding to eIF4A and Mnk1

The X-ray structure of the C-terminal region of human eukaryotic translation initiation factor 4G (eIF4G) has been determined at 2.2 A resolution, revealing two atypical HEAT-repeat domains. eIF4G recruits various translation factors and the 40S ribosomal subunit to the mRNA 5' end. In higher eukaryotes, the C terminus of eIF4G (4G/C) supports translational regulation by recruiting eIF4A, an RNA helicase, and Mnk1, the kinase responsible for phosphorylating eIF4E. Structure-guided surface mutagenesis and protein-protein interaction assays were used to identify binding sites for eIF4A and Mnk1 within the HEAT-repeats of 4G/C. p97/DAP5, a translational modulator homologous to eIF4G, lacks an eIF4A binding site in the corresponding region. The second atypical HEAT domain of the 4G/C binds Mnk1 using two conserved aromatic/acidic-box (AA-box) motifs. Within the first AA-box, the aromatic residues contribute to the hydrophobic core of the domain, while the acidic residues form a negatively charged surface feature suitable for electrostatic interactions with basic residues in Mnk1.     

A novel function of the MA-3 domains in transformation and translation suppressor Pdcd4 is essential for its binding to eukaryotic translation initiation factor 4A

An alpha-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4(E249K), Pdcd4(D253A), Pdcd4(D414K), Pdcd4(D418A), Pdcd4(E249K,D414K), and Pdcd4(D253A,D418A)) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4(D253A) and Pdcd4(D253A,D418A) do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4(D253A), Pdcd4(D418A), or Pdcd4(D235A,D418A). Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.     

Arabidopsis nonresponding to oxylipins locus NOXY7 encodes a yeast GCN1 homolog that mediates noncanonical translation regulation and stress adaptation

Stress adaptation and translational regulation was studied using noxy7 (nonresponding to oxylipins7) from a series of Arabidopsis thaliana mutants. We identified the noxy7 mutation in At1g64790, which encodes a homolog of the yeast translational regulator General Control Nonderepressible1 (GCN1) that activates the GCN2 kinase; GCN2 in turn phosphorylates the α subunit of the translation initiation factor eIF2. This regulatory circuit is conserved in yeast and mammals, in which phosphorylated eIF2α (P‐eIF2α) facilitates stress adaptation by inhibiting protein synthesis. In phenotypic and de novo protein synthesis studies with Arabidopsis mutants, we found that NOXY7/GCN1 and GCN2 mediate P‐eIF2α formation and adaptation to amino acid deprivation; however, P‐eIF2α formation is not linked to general protein synthesis arrest. Additional evidence suggested that NOXY7/GCN1 but not GCN2 regulates adaptation to mitochondrial dysfunction, high boron concentration, and activation of plant immunity to infection by Pseudomonas syringae pv tomato (Pst). In these responses, NOXY7/GCN1 acts with GCN20 to regulate translation in a noncanonical pathway independently of GCN2 and P‐eIF2α. These results show the lesser functional relevance of GCN2 and P‐eIF2α in plants relative to other eukaryotes and highlight the prominent role of NOXY7/GCN1 and GCN20 in regulation of translation and stress adaptation in plants.     

Evolutionarily conserved non-AUG translation initiation in NAT1/p97/DAP5 (EIF4G2)

Only a few cases of exclusive translation initiation at non-AUG codons have been reported. We recently demonstrated that mammalian NAT1 mRNA, encoded by EIF4G2, uses GUG as its only translation initiation codon. In this study, we identified NAT1 orthologs from chicken, Xenopus, and zebrafish and found that in all species, the GUG codon also serves as the initiation codon. In all species, the GUG codon fulfilled the reported requirements for non-AUG initiation: an optimal Kozak motif and a downstream hairpin structure. Site-directed mutagenesis showed that nucleotides at positions -3 and +4 are critical for the GUG-mediated translation initiation in vitro. We found that NAT1 orthologs in Drosophila melanogaster and Halocynthia roretzi also use non-AUG start codons, demonstrating evolutionary conservation of the noncanonical translation initiation.     

Mextli Is a Novel Eukaryotic Translation Initiation Factor 4E-Binding Protein That Promotes Translation in Drosophila melanogaster

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.     

Distinct regulation of internal ribosome entry site-mediated translation following cellular stress is mediated by apoptotic fragments of eIF4G translation initiation factor family members eIF4GI and p97/DAP5/NAT1

Many cellular stresses lead to the inhibition of protein synthesis. Despite this, some cellular mRNAs are selectively translated under these conditions. It was suggested that the presence of internal ribosome entry site (IRES) sequences in the 5'-untranslated regions allow these mRNAs to be actively translated despite the overall cessation of protein synthesis. Here we tested the hypothesis that the IRES elements of genes that are involved in the control of cell survival are distinctly regulated by cellular stresses. We show that the transient conditions of cellular stress favor the translation of pro-survival IRES, while the severe apoptotic conditions support translation of pro-death IRES elements. Furthermore, activation of pro-death IRES during the etoposide-induced apoptosis is caspase-dependent and correlates with the expression of apoptotic fragments of two members of the eIF4G translation initiation factor family, p97/DAP5/NAT1 and eIF4GI. Our results suggest that the regulation of IRES translation during stress contributes to the fine-tuning of cell fate.     

The first large-scale nucleic acid amplification testing (NAT) of donated blood using multiplex reagent for simultaneous detection of HBV, HCV, and HIV-1 and significance of NAT for HBV.

The first nationwide nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type 1 (HIV-1) of voluntarily donated blood after serological pre-screening and before release of cellular components and plasma for fractionation was implemented by the Japanese Red Cross Blood Transfusion Services. From February 1, 2000 to April 30, 2001, specimens from 6,805,010 units of serologically negative donation were screened in minipools of 50 samples within 24 hr after blood donation by NAT using multiplex HBV/HCV/HIV-1 reagent for blood transfusion including short shelf-life platelets. Among them, 112 HBV DNA-positives, 25 HCV RNA positives and 4 HIV-1 RNA positives were screened out and we could prevent transfusion of these NAT positive units. Subtypes/genotypes of HBV DNA, adr/C, adw/A, adw/B, adw/C, ayr/C and ayw/D were found and adr/C was predominant. A total of 61.6 % of them (69/112) were negative by overnight EIA. Sixth three of HBV NAT-positive samples carried virus loads less than 10(4) copies/mL and 92.1 % of them (58/63) were negative by overnight EIA. The virus growth curves of HBV in 6 cases obtained by retrospective and prospective follow-up study showed exponential straight lines in the early stage of serological window periods and the log times of HBV growth (10 fold increase) in serological window period were between 4.6 and 7.6 days. NAT screening with highly sensitive reagents in pool of specimens is useful to exclude blood units with low level of HBV and HBV mutants from blood transfusion.     

Eukaryotic translation initiation factor 5 is critical for integrity of the scanning preinitiation complex and accurate control of GCN4 translation

The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal pre-initiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for pre-initiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts- phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd- phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNA(i)Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn- phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the post-assembly process in the 48S complex, likely during the scanning process.     

Identification and characterization of the expression of the translation initiation factor 4A (eIF4A) from Drosophila melanogaster

We have identified the initiation factor 4A (eIF4A) in a two-dimensional protein database of Drosophila wing imaginal discs. eIF4A, a member of the DEAD-box family of RNA helicases, forms the active eIF4F complex that in the presence of eIF4B and eIF4H unwinds the secondary structure of the 5'-UTR of mRNAs during translational initiation. Two-dimensional gel electrophoresis and microsequencing allowed us to purify eIF4A, and generate specific polyclonal antibodies. A combination of immunoblotting and labelling with [(35)S]methionine + [(35)S]cysteine revealed the existence of a single eIF4A isoform encoded by a previously reported gene that maps to chromosome 2L at 26A7-9. Expression of this gene yields two mRNA species, generated by alternative splicing in the 3'-untranslated region. The two mRNAs contain the same open reading frame and produce the identical eIF4A protein. No expression was detected of the eIF4A-related gene CG7483. We detected eIF4A protein expression in the wing imaginal discs of several Drosophila species, and in haltere, leg 1, leg 2, leg 3, and eye-antenna imaginal discs of D. melanogaster. Examination of eIF4A in tumor suppressor mutants showed significantly increased (> 50%) expression in the wing imaginal discs of these larvae. We observed ubiquitous expression of eIF4A mRNA and protein during Drosophila embryogenesis. Yeast two-hybrid analysis demonstrated the in vivo interaction of Drosophila eIF4G with the N-terminal third of eIF4A.     

Diet-dependent effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via eIF4E

The control of cellular growth is tightly linked to the regulation of protein synthesis. A key function in translation initiation is fulfilled by the 5' cap binding eukaryotic initiation factor 4E (eIF4E), and dysregulation of eIF4E is associated with malignant transformation and tumorigenesis . In mammals, the activity of eIF4E is modulated by phosphorylation at Ser209 by mitogen-activated protein kinases (MAPK)-interacting kinases 1 and 2 (Mnk1 and Mnk2) , which themselves are activated by ERK and p38 MAPK in response to mitogens, cytokines or cellular stress . Whether phosphorylation of eIF4E at Ser209 exerts a positive or inhibitory effect on translation efficiency has remained controversial. Here we provide a genetic characterization of the Drosophila homolog of Mnk1/2, Lk6. Lk6 function is dispensable under a high protein diet, consistent with the recent finding that mice lacking both Mnk1 and Mnk2 are not growth-impaired . Interestingly, loss of Lk6 function causes a significant growth reduction when the amino acid content in the diet is reduced. Overexpression of Lk6 also results in growth inhibition in an eIF4E-dependent manner. We propose a model of eIF4E regulation that may reconcile the contradictory findings with regard to the role of phosphorylation by Mnk1/2.     

Drosophila Lk6 kinase controls phosphorylation of eukaryotic translation initiation factor 4E and promotes normal growth and development

Eukaryotic initiation factor 4E (eIF4E) controls a crucial step of translation initiation and is critical for cell growth . Biochemical studies have shown that it undergoes a regulated phosphorylation by the MAP-kinase signal-integrating kinases Mnk1 and Mnk2 . Although the role of eIF4E phosphorylation in mammalian cells has remained elusive , recent work in Drosophila has established that it is required for growth and development . Here, we demonstrate that a previously identified Drosophila kinase called Lk6 is the functional homolog of mammalian Mnk kinases. We generated lk6 loss-of-function alleles and found that eIF4E phosphorylation is dramatically reduced in lk6 mutants. Importantly, lk6 mutants exhibit reduced viability, slower development, and reduced adult size, demonstrating that Lk6 function is required for organismal growth. Moreover, we show that uniform lk6 expression rescues the lethality of eIF4E hypomorphic mutants in an eIF4E phosphorylation site-dependent manner and that the two proteins participate in a common complex in Drosophila S2 cells, confirming the functional link between Lk6 and eIF4E. This work demonstrates that Lk6 exerts a tight control on eIF4E phosphorylation and is necessary for normal growth and development.     

The translational inhibitor 4E-BP is an effector of PI(3)K/Akt signalling and cell growth in Drosophila

The initiation factor 4E for eukaryotic translation (eIF4E) binds the messenger RNA 5'-cap structure and is important in the regulation of protein synthesis. Mammalian eIF4E activity is inhibited when the initiation factor binds to the translational repressors, the 4E-binding proteins (4E-BPS). Here we show that the Drosophila melanogaster 4E-BP (d4E-BP) is a downstream target of the phosphatidylinositol-3-OH kinase (PI(3)K) signal-transduction cascade, which affects the interaction of d4E-BP with eIF4E. Ectopic expression of a highly active d4E-BP mutant in wing-imaginal discs causes a reduction of wing size, brought about by a decrease in cell size and number. A marked reduction in cell size was also observed in post-mitotic cells. Expression of d4E-BP in the eye and wing together with PI(3)K or dAkt1, the serine/threonine kinase downstream of PI(3)K, resulted in suppression of the growth phenotype elicited by these kinases. Our results support a role for d4E-BP as an effector of cell growth.