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Photosynthesis requires chloroplasts, in which most proteins are nucleus-encoded and produced via cytoplasmic translation. The translation initiation factor eIF5B gates the transition from initiation (I) to elongation (E), and the Kozak motif is associated with translation efficiency, but their relationship is previously unknown. Here, with ribosome profiling, we determined the genome-wide I-E transition efficiencies. We discovered that the most prevalent Kozak motif is associated with high I-E transition efficiency in Arabidopsis, rice, and wheat, thus implicating the potential of the Kozak motif in facilitating the I-E transition. Indeed, the effects of Kozak motifs in promoting translation depend on HOT3/eIF5B1 in Arabidopsis. HOT3 preferentially promotes the translation of photosynthesis-associated nuclear genes in a Kozak motif-dependent manner, which explains the chloroplast defects and reduced photosynthesis activity of hot3 mutants. Our study linked the Kozak motif to eIF5B-mediated I-E transition during translation and uncovered the function of HOT3 in the cytoplasmic translational control of chloroplast biogenesis and photosynthesis., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The phosphorylation of eukaryotic translational initiation factors has been shown to play a significant role in controlling the synthesis of protein. Viral infection, environmental stress, and growth circumstances cause phosphorylation or dephosphorylation of plant initiation factors. Our findings indicate that casein kinase 2 can phosphorylate recombinant wheat eIFiso4E and eIFiso4G generated from E. coli in vitro. For wheat eIFiso4E, Ser-207 was found to be the in vitro phosphorylation site. eIFiso4E lacks an amino acid that can be phosphorylated at the position corresponding to Ser-209, the phosphorylation site in mammalian eIF4E, yet phosphorylation of eIFiso4E has effects on VPg binding affinity that are similar to those of phosphorylation of mammalian eIF4E. The addition of VPg and phosphorylated eIFiso4F to depleted wheat germ extract (WGE) leads to enhancement of translation of both uncapped and capped viral mRNA. The addition of PABP together with eIFiso4Fp and eIF4B to depleted WGE increases both uncapped and capped mRNA translation. However, it exhibits a translational advantage specifically for uncapped mRNA, implying that the phosphorylation of eIFiso4F hinders cap binding while promoting VPg binding, thereby facilitating uncapped translation. These findings indicate TEV virus mediates VPg-dependent translation by engaging a mechanism entailing phosphorylated eIFiso4Fp and PABP. To elucidate the molecular mechanisms underlying these observed effects, we studied the impact of PABP and/or eIF4B on the binding of VPg with eIFiso4Fp. The inclusion of PABP and eIF4B with eIFiso4Fp resulted in about 2-fold increase in affinity for VPg (Kd = 24 ± 1.7 nM), as compared to the affinity of eIFiso4Fp alone (Kd = 41.0 ± 3.1 nM). The interactions between VPg and eIFiso4Fp were determined to be both enthalpically and entropically favorable, with the enthalpic contribution accounting for 76-97% of the ΔG at 25°C, indicating a substantial role of hydrogen bonding in enhancing the stability of the complex. The binding of PABP to eIFiso4Fp·4B resulted in a conformational alteration, leading to a significant enhancement in the binding affinity to VPg. These observations suggest PABP enhances the affinity between eIFiso4Fp and VPg, leading to an overall conformational change that provides a stable platform for efficient viral translation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

During translation, a stop codon on the mRNA signals the ribosomes to terminate the process. In certain mRNAs, the termination fails due to the recoding of the canonical stop codon, and ribosomes continue translation to generate C-terminally extended protein. This process, termed stop codon readthrough (SCR), regulates several cellular functions. SCR is driven by elements/factors that act immediately downstream of the stop codon. Here, we have analysed the process of SCR using a simple mathematical model to investigate how the kinetics of translating ribosomes influences the efficiency of SCR. Surprisingly, the analysis revealed that the rate of translation inversely regulates the efficiency of SCR. We tested this prediction experimentally in mammalian AGO1 and MTCH2 mRNAs. Reduction in translation either globally by harringtonine or locally by rare codons caused an increase in the efficiency of SCR. Thus, our study has revealed a hitherto unknown mode of regulation of SCR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

In tumourigenesis, oncogenes or dysregulated tumour suppressor genes alter the canonical translation machinery leading to a reprogramming of the translatome that, in turn, promotes the translation of selected mRNAs encoding proteins involved in proliferation and metastasis. It is therefore unsurprising that abnormal expression levels and activities of eukaryotic initiation factors (eIFs), elongation factors (eEFs) or termination factors (eRFs) are associated with poor outcome for patients with a wide range of cancers. In this review we discuss how RNA binding proteins (RBPs) within the canonical translation factor machinery are dysregulated in cancers and how targeting such proteins is leading to new therapeutic avenues., Competing Interests: Conflict of interest statement The authors declare that there are no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Local translation can provide a rapid, spatially targeted supply of new proteins in distal dendrites to support synaptic changes that underlie learning. Learning and memory are especially sensitive to manipulations of translational control mechanisms, particularly those that target the initiation step, and translation initiation at synapses could be a means of maintaining synapse specificity during plasticity. Initiation predominantly occurs via recruitment of ribosomes to the 5' mRNA cap by complexes of eukaryotic initiation factors (eIFs), and the interaction between eIF4E and eIF4G1 is a particularly important target of translational control pathways. Pharmacological inhibition of eIF4E-eIF4G1 binding impairs formation of memory for aversive Pavlovian conditioning as well as the accompanying increase in polyribosomes in the heads of dendritic spines in the lateral amygdala (LA). This is consistent with a role for initiation at synapses in memory formation, but whether eIFs are even present near synapses is unknown. To determine whether dendritic spines contain eIFs and whether eIF distribution is affected by learning, we combined immunolabeling with serial section transmission electron microscopy (ssTEM) volume reconstructions of LA dendrites after Pavlovian conditioning. Labeling for eIF4E, eIF4G1, and eIF2α-another key target of regulation-occurred in roughly half of dendritic spines, but learning effects were only found for eIF4E, which was upregulated in the heads of dendritic spines. Our results support the possibility of regulated translation initiation as a means of synapse-specific protein targeting during learning and are consistent with the model of eIF4E availability as a central point of control., (© 2021 Wiley Periodicals LLC.)

Effective pharmacotherapy for major depressive disorder remains a major challenge, as more than 30% of patients are resistant to the first line of treatment (selective serotonin reuptake inhibitors) 1 . Sub-anaesthetic doses of ketamine, a non-competitive N-methyl-D-aspartate receptor antagonist 2,3 , provide rapid and long-lasting antidepressant effects in these patients 4-6 , but the molecular mechanism of these effects remains unclear 7,8 . Ketamine has been proposed to exert its antidepressant effects through its metabolite (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) 9 . The antidepressant effects of ketamine and (2R,6R)-HNK in rodents require activation of the mTORC1 kinase 10,11 . mTORC1 controls various neuronal functions 12 , particularly through cap-dependent initiation of mRNA translation via the phosphorylation and inactivation of eukaryotic initiation factor 4E-binding proteins (4E-BPs) 13 . Here we show that 4E-BP1 and 4E-BP2 are key effectors of the antidepressant activity of ketamine and (2R,6R)-HNK, and that ketamine-induced hippocampal synaptic plasticity depends on 4E-BP2 and, to a lesser extent, 4E-BP1. It has been hypothesized that ketamine activates mTORC1-4E-BP signalling in pyramidal excitatory cells of the cortex 8,14 . To test this hypothesis, we studied the behavioural response to ketamine and (2R,6R)-HNK in mice lacking 4E-BPs in either excitatory or inhibitory neurons. The antidepressant activity of the drugs is mediated by 4E-BP2 in excitatory neurons, and 4E-BP1 and 4E-BP2 in inhibitory neurons. Notably, genetic deletion of 4E-BP2 in inhibitory neurons induced a reduction in baseline immobility in the forced swim test, mimicking an antidepressant effect. Deletion of 4E-BP2 specifically in inhibitory neurons also prevented the ketamine-induced increase in hippocampal excitatory neurotransmission, and this effect concurred with the inability of ketamine to induce a long-lasting decrease in inhibitory neurotransmission. Overall, our data show that 4E-BPs are central to the antidepressant activity of ketamine.

The 5' untranslated region (5'UTR) is critical in determining post-transcriptional control, which is partly mediated by short upstream open reading frames (uORFs) present in half of mammalian transcripts. uORFs are generally considered to provide functionally important repression of the main-ORF by engaging initiating ribosomes, but under specific environmental conditions such as cellular stress, uORFs can become essential to activate the translation of the main coding sequence. In addition, a growing number of uORF-encoded bioactive microproteins have been described, which have the potential to significantly increase cellular protein diversity. Here we review the diverse cellular contexts in which uORFs play a critical role and discuss the molecular mechanisms underlying their function and regulation. The progress over the last decades in dissecting uORF function suggests that the 5'UTR remains an exciting frontier towards understanding how the cellular proteome is shaped in health and disease., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Translation efficiency varies considerably between different mRNAs, thereby impacting protein expression. Translation of the stress response master-regulator ATF4 increases upon stress, but the molecular mechanisms are not well understood. We discover here that translation factors DENR, MCTS1 and eIF2D are required to induce ATF4 translation upon stress by promoting translation reinitiation in the ATF4 5'UTR. We find DENR and MCTS1 are only needed for reinitiation after upstream Open Reading Frames (uORFs) containing certain penultimate codons, perhaps because DENR•MCTS1 are needed to evict only certain tRNAs from post-termination 40S ribosomes. This provides a model for how DENR and MCTS1 promote translation reinitiation. Cancer cells, which are exposed to many stresses, require ATF4 for survival and proliferation. We find a strong correlation between DENR•MCTS1 expression and ATF4 activity across cancers. Furthermore, additional oncogenes including a-Raf, c-Raf and Cdk4 have long uORFs and are translated in a DENR•MCTS1 dependent manner.

The Integrated Stress Response (ISR) helps metazoan cells adapt to cellular stress by limiting the availability of initiator methionyl-tRNA for translation. Such limiting conditions paradoxically stimulate the translation of ATF4 mRNA through a regulatory 5' leader sequence with multiple upstream Open Reading Frames (uORFs), thereby activating stress-responsive gene expression. Here, we report the identification of two critical regulators of such ATF4 induction, the noncanonical initiation factors eIF2D and DENR. Loss of eIF2D and DENR in Drosophila results in increased vulnerability to amino acid deprivation, susceptibility to retinal degeneration caused by endoplasmic reticulum (ER) stress, and developmental defects similar to ATF4 mutants. eIF2D requires its RNA-binding motif for regulation of 5' leader-mediated ATF4 translation. Consistently, eIF2D and DENR deficient human cells show impaired ATF4 protein induction in response to ER stress. Altogether, our findings indicate that eIF2D and DENR are critical mediators of ATF4 translational induction and stress responses in vivo.

Eukaryotic translation initiation factor 6 (eIF6) is essential for the synthesis of 60S ribosomal subunits and for regulating the association of 60S and 40S subunits. A mechanistic understanding of how eIF6 modulates translation in response to stress, specifically starvation-induced stress, is lacking. We here show a novel mode of eIF6 regulation by glycogen synthase kinase 3 (GSK3) that is predominantly active in response to serum starvation. Both GSK3α and GSK3β phosphorylate human eIF6. Multiple residues in the C terminus of eIF6 are phosphorylated by GSK3 in a sequential manner. In response to serum starvation, eIF6 accumulates in the cytoplasm, and this altered localization depends on phosphorylation by GSK3. Disruption of eIF6 phosphorylation exacerbates the translation inhibitory response to serum starvation and stalls cell growth. These results suggest that eIF6 regulation by GSK3 contributes to the attenuation of global protein synthesis that is critical for adaptation to starvation-induced stress., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Jungers et al.)

Activation of the immune costimulatory molecule cluster of differentiation 40 (CD40) in Müller glia has been implicated in the initiation of diabetes-induced retinal inflammation. Results from previous studies support that CD40 protein expression is elevated in Müller glia of diabetic mice; however, the mechanisms responsible for this increase have not been explored. Here, we evaluated the hypothesis that diabetes augments translation of the Cd40 mRNA. Mice receiving thiamet G (TMG), an inhibitor of the O -GlcNAc hydrolase O -GlcNAcase, exhibited enhanced retinal protein O -GlcNAcylation and increased Cd40 mRNA translation. TMG administration also promoted Cd40 mRNA association with Müller cell-specific ribosomes isolated from the retina of RiboTag mice. Similar effects on O -GlcNAcylation and Cd40 mRNA translation were also observed in the retina of a mouse model of type 1 diabetes. In cultured cells, TMG promoted sequestration of the cap-binding protein eIF4E (eukaryotic translation in initiation factor 4E) by 4E-BP1 (eIF4E-binding protein 1) and enhanced cap-independent Cd40 mRNA translation as assessed by a bicistronic reporter that contained the 5'-UTR of the Cd40 mRNA. Ablation of 4E-BP1/2 prevented the increase in Cd40 mRNA translation in TMG-exposed cells, and expression of a 4E-BP1 variant that constitutively sequesters eIF4E promoted reporter activity. Extending on the cell culture results, we found that in contrast to WT mice, diabetic 4E-BP1/2-deficient mice did not exhibit enhanced retinal Cd40 mRNA translation and failed to up-regulate expression of the inflammatory marker nitric-oxide synthase 2. These findings support a model wherein diabetes-induced O -GlcNAcylation of 4E-BP1 promotes Cd40 mRNA translation in Müller glia., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Dierschke et al.)

Mitochondrial genomes code for several core components of respiratory chain complexes. Thus, mitochondrial translation is of great importance for the organelle as well as for the whole cell. In yeast, mitochondrial translation initiation factor 3, Aim23p, is not essential for the organellar protein synthesis; however, its absence leads to a significant quantitative imbalance of the mitochondrial translation products. This fact points to a possible specific action of Aim23p on the biosynthesis of some mitochondrial protein species. In this work, we examined such peculiar effects of Aim23p in relation to yeast mitochondrial COX2 mRNA translation. We show that Aim23p is indispensable to this process. According to our data, this is mediated by Aimp23p interaction with the known specific factor of the COX2 mRNA translation, Pet111p. If there is no Aim23p in the yeast cells, an increased amount of Pet111p ensures proper COX2 mRNA translation. Our results demonstrate the additional non-canonical function of initiation factor 3 in yeast mitochondrial translation.

The initiation of protein synthesis in bacteria is ruled by three canonical factors: IF1, IF2, and IF3. This system persists in human mitochondria; however, it functions in a rather different way due to specialization and adaptation to the organellar micro-environment. We focused on human mitochondrial IF3, which was earlier studied in vitro, but no knock-out cellular models have been published up to date. In this work, we generated human HeLa cell lines deficient in the MTIF3 gene and analyzed their mitochondrial function. Despite the lack of IF3mt in these cells, they preserved functional mitochondria capable of oxygen consumption and protein synthesis; however, the translation of ATP6 mRNA was selectively decreased which compromised the assembly of ATP synthase. Together with the analogous results obtained earlier for baker's yeast mitochondrial IF3, our findings point to a functional divergence of mitochondrial initiation factors from their bacterial ancestors.

Translation is a fundamental step in gene expression that regulates multiple developmental and stress responses. One key step of translation initiation is the association between eIF4E and eIF4G. This process is regulated in different eukaryotes by proteins that bind to eIF4E; however, evidence of eIF4E-interacting proteins able to regulate translation is missing in plants. Here, we report the discovery of CERES, a plant eIF4E-interacting protein. CERES contains an LRR domain and a canonical eIF4E-binding site. Although the CERES-eIF4E complex does not include eIF4G, CERES forms part of cap-binding complexes, interacts with eIF4A, PABP and eIF3, and co-sediments with translation initiation complexes in vivo. Moreover, CERES promotes translation in vitro and general translation in vivo, while it modulates the translation of specific mRNAs related to light and carbohydrate response. These data suggest that CERES is a non-canonical translation initiation factor that modulates translation in plants.

Purpose: We reported previously that retinas of mice with inherited retinal degeneration make less protein than retinas of normal mice. Despite recent studies suggesting that diminished protein synthesis rates may contribute to neurologic disorders, a direct link between protein synthesis rates and the progression of neurodegeneration has not been established. Moreover, it remains unclear whether reduced protein synthesis could be involved in retinal pathogenesis. Dysregulation of AKT/mTOR signaling has been reported in the retina during retinal degeneration, but to what extent this signaling contributes to translational attenuation in these mice remains uncertain., Methods: C57BL/6J and rd16 mice were subcutaneously injected with anisomycin to chronically inhibit protein synthesis rates. An AAV2 construct encoding constitutively active 4ebp1 was subretinally delivered in wildtype animals to lower protein synthesis rates. 4ebp1/2 were knocked out in rd16 mice., Results: Anisomycin treatment lowered retinal translation rates, accelerated retinal degeneration in rd16 mice, and initiated cell death in the retinas of C57BL/6J mice. AAV-mediated transfer of constitutively active 4ebp1-4A into the subretinal space of wildtype animals inhibited protein synthesis, and led to reduced electroretinography amplitudes and fewer ONL nuclei. Finally, we report that restoring protein synthesis rates by knocking out 4ebp1/2 was associated with an approximately 2-fold increase in rhodopsin levels and a delay in retinal degeneration in rd16 mice., Conclusions: Our study indicates that protein synthesis inhibition is likely not a cell defense mechanism in the retina by which deteriorating photoreceptors survive, but may be harmful to degenerating retinas, and that restoring protein synthesis may have therapeutic potential in delaying the progression of retinal degeneration.

Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of select soluble proteins. Nearly one-third of the soluble proteins are predicted to be recognized by this pathway, yet only a minor fraction of this proteome has been identified as CMA substrates in cancer cells. Here, we undertook a quantitative multiplex mass spectrometry approach to study the proteome of isolated lysosomes in cancer cells during CMA-activated conditions. By integrating bioinformatics analyses, we identified and categorized proteins of multiple cellular pathways that were specifically targeted by CMA. Beyond verifying metabolic pathways, we show that multiple components involved in select biological processes, including cellular translation, was specifically targeted for degradation by CMA. In particular, several proteins of the translation initiation complex were identified as bona fide CMA substrates in multiple cancer cell lines of distinct origin and we show that CMA suppresses cellular translation. We further show that the identified CMA substrates display high expression in multiple primary cancers compared to their normal counterparts. Combined, these findings uncover cellular processes affected by CMA and reveal a new role for CMA in the control of translation in cancer cells. Abbreviations: 6-AN: 6-aminonicotinamide; ACTB: actin beta; AR7: atypical retinoid 7; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; CTS: cathepsins; DDX3X: DEAD-box helicase 3 X-linked; EEF2: eukaryotic translation elongation factor 2; EIF4A1: eukaryotic translation initiation factor 4A1; EIF4H: eukaryotic translation initiation factor 4H; GEO: Gene Expression Omnibus; GO: Gene Ontology; GSEA: gene set enrichment analysis; HK2: hexokinase 2; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; LAMP: lysosomal-associated membrane protein; LDHA: lactate dehydrogenase A; NES: normalized enrichment score; NFKBIA: NFKB inhibitor alpha; PCA: principle component analysis; PQ: paraquat; S.D.: standard deviation; SUnSET: surface sensing of translation; TMT: tandem mass tags; TOMM40/TOM40: translocase of outer mitochondrial membrane 40.

Translational readthrough generates proteins with extended C-termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let-7a miRNA-binding site, and readthrough is promoted by let-7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post-transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Sarcopenia is the loss of muscle mass/function that occurs during the aging process. The links between mechanistic target of rapamycin (mTOR) activity and muscle development are largely documented, but the role of its downstream targets in the development of sarcopenia is poorly understood. Eukaryotic initiation factor 4E-binding proteins (4E-BPs) are targets of mTOR that repress mRNA translation initiation and are involved in the control of several physiological processes. However, their role in skeletal muscle is still poorly understood. The goal of this study was to assess how loss of 4E-BP1 and 4E-BP2 expression impacts skeletal muscle function and homeostasis in aged mice and to characterize the associated metabolic changes by metabolomic and lipidomic profiling., Methods: Twenty-four-month-old wild-type and whole body 4E-BP1/4E-BP2 double knockout (DKO) mice were used to measure muscle mass and function. Protein homeostasis was measured ex vivo in extensor digitorum longus by incorporation of l-[U- 14 C]phenylalanine, and metabolomic and lipidomic profiling of skeletal muscle was performed by Metabolon, Inc., Results: The 4E-BP1/2 DKO mice exhibited an increase in muscle mass that was associated with increased grip strength (P < 0.05). Protein synthesis was higher under both basal (+102%, P < 0.05) and stimulated conditions (+65%, P < 0.05) in DKO skeletal muscle. Metabolomic and complex lipid analysis of skeletal muscle revealed robust differences pertaining to amino acid homeostasis, carbohydrate abundance, and certain aspects of lipid metabolism. In particular, levels of most free amino acids were lower within the 4E-BP1/2 DKO muscle. Interestingly, although glucose levels were unchanged, differences were observed in the isobaric compound maltitol/lactitol (33-fold increase, P < 0.01) and in several additional carbohydrate compounds. 4E-BP1/2 depletion also resulted in accumulation of medium-chain acylcarnitines and a 20% lower C2/C0 acylcarnitine ratio (P < 0.01) indicative of reduced β-oxidation., Conclusions: Taken together, these findings demonstrate that deletion of 4E-BPs is associated with perturbed energy metabolism in skeletal muscle and could have beneficial effects on skeletal muscle mass and function in aging mice. They also identify 4E-BPs as potential targets for the treatment of sarcopenia., (© 2019 The Authors. Journal of Cachexia, Sarcopenia and Muscle published by John Wiley & Sons Ltd on behalf of the Society on Sarcopenia, Cachexia and Wasting Disorders.)

Altered lipid metabolism and aberrant protein translation are strongly associated with cancerous outgrowth; however, the inter-regulation of these key processes is still underexplored in diffuse large B-cell lymphoma (DLBCL). Although fatty acid synthase (FASN) activity is reported to positively correlate with PI3K-Akt-mTOR pathway that can modulate protein synthesis, the precise impact of FASN inhibition on this process is still unknown. Herein, we demonstrate that attenuating FASN expression or its activity significantly reduces eIF4B (eukaryotic initiation factor 4B) levels and consequently overall protein translation. Through biochemical studies, we identified eIF4B as a bonafide substrate of USP11, which stabilizes and enhances eIF4B activity. Employing both pharmacological and genetic approaches, we establish that FASN-induced PI3K-S6Kinase signaling phosphorylates USP11 enhancing its interaction with eIF4B and thereby promoting oncogenic translation.

Controlling translation initiation is an efficient way to regulate gene expression at the post-transcriptional level. However, current knowledge regarding regulatory proteins and their modes of controlling translation initiation is still limited. In this study, we employed tandem affinity purification and mass spectrometry to screen for unknown proteins associated with the translation initiation machinery. Ubiquitin specific peptidase 9, X-linked (USP9X), was identified as a novel binding partner, that interacts with the eukaryotic translation initiation factor 4B (eIF4B) in a mRNA-independent manner. USP9X-deficient cells presented significantly impaired nascent protein synthesis, cap-dependent translation initiation and cellular proliferation. USP9X can selectively alter the translation of pro-oncogenic mRNAs, such as c-Myc and XIAP. Moreover, we found that eIF4A1, which is primarily ubiquitinated at Lys-369, is the substrate of USP9X. USP9X dysfunction increases the ubiquitination of eIF4A1 and enhances its degradation. Our results provide evidence that USP9X is a novel regulator of the translation initiation process via deubiquitination of eIF4A1, which offers new insight in understanding the pivotal role of USP9X in human malignancies and neurodevelopmental disorders., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA-protein interactions play a pivotal role in the function of picornavirus internal ribosome entry site (IRES) elements. Here we analysed the impact of Ras GTPase SH3 domain binding protein 1 (G3BP1) in the IRES activity of foot-and-mouth disease virus (FMDV). We found that G3BP1 interacts directly with three distinct sequences of the IRES element using RNA electrophoretic mobility-shift assays. Analysis of the interaction with domain 5 indicated that the G3BP1 binding-site is placed at the single-stranded region although it allows large sequence heterogeneity and the hairpin located upstream of this region enhances retarded complex formation. In addition, G3BP1 interacts directly with the polypyrimidine tract-binding protein and the translation initiation factor 4B (eIF4B) through the C-terminal region. Moreover, G3BP1 is cleaved during FMDV infection yielding two fragments, Ct-G3BP1 and Nt-G3BP1. Both fragments inhibit cap- and IRES-dependent translation, but the Ct-G3BP1 fragment shows a stronger effect on IRES-dependent translation. Assembly of complexes with G3BP1 results in a significantly reduced local flexibility of the IRES element, consistent with the negative effect of this protein. Our results highlight the IRES-binding capacity of G3BP1 and illustrate its function as a translation inhibitor., (© 2017 Federation of European Biochemical Societies.)

The conserved eukaryotic translation initiation factor 5B, eIF5B, is a GTPase that acts late in translation initiation. We found that an Arabidopsis thaliana mutant sensitive to hot temperatures 3 ( hot3-1 ), which behaves as the wild type in the absence of stress but is unable to acclimate to high temperature, carries a missense mutation in the eIF5B1 gene (At1g76810), producing a temperature sensitive protein. A more severe, T-DNA insertion allele ( hot3-2 ) causes pleiotropic developmental phenotypes. Surprisingly, Arabidopsis has three other eIF5B genes that do not substitute for eIF5B1 ; two of these appear to be in the process of pseudogenization. Polysome profiling and RNA-seq analysis of hot3-1 plants show delayed recovery of polysomes after heat stress and reduced translational efficiency (TE) of a subset of stress protective proteins, demonstrating the critical role of translational control early in heat acclimation. Plants carrying the severe hot3-2 allele show decreased TE of auxin-regulated, ribosome-related, and electron transport genes, even under optimal growth conditions. The hot3-2 data suggest that disrupting specific eIF5B interactions on the ribosome can, directly or indirectly, differentially affect translation. Thus, modulating eIF5B interactions could be another mechanism of gene-specific translational control., (© 2017 American Society of Plant Biologists. All rights reserved.)

Recent technological advancements have shown tremendous mechanistic accomplishments in our understanding of the mechanism of messenger RNA translation in eukaryotic cells. Eukaryotic messenger RNA translation is very complex process that includes four phases (initiation, elongation, termination, and ribosome recycling) and diverse mechanisms involving protein and non-protein molecules. Translation regulation is principally achieved during initiation step of translation, which is organized by multiple eukaryotic translation initiation factors. Eukaryotic translation initiation factor proteins help in stabilizing the formation of the functional ribosome around the start codon and provide regulatory mechanisms in translation initiation. Dysregulated messenger RNA translation is a common feature of tumorigenesis. Various oncogenic and tumor suppressive genes affect/are affected by the translation machinery, making the components of the translation apparatus promising therapeutic targets for the novel anticancer drug. This review provides details on the role of eukaryotic translation initiation factors in messenger RNA translation initiation, their contribution to onset and progression of tumor, and how dysregulated eukaryotic translation initiation factors can be used as a target to treat carcinogenesis.

Barley yellow dwarf virus RNA, lacking a 5' cap and a 3' poly(A) tail, contains a cap-independent translation element (BTE) in the 3'-untranslated region that interacts with host translation initiation factor eIF4G. To determine how eIF4G recruits the mRNA, three eIF4G deletion mutants were constructed: (i) eIF4G601-1196, containing amino acids 601-1196, including the putative BTE-binding region, and binding domains for eIF4E, eIF4A, and eIF4B; (ii) eIF4G601-1488, which contains an additional C-terminal eIF4A-binding domain; and (iii) eIF4G742-1196, which lacks the eIF4E-binding site. eIF4G601-1196 binds BTE tightly and supports efficient translation. The helicase complex, consisting of eIF4A, eIF4B, and ATP, stimulated BTE binding with eIF4G601-1196 but not eIF4G601-1488, suggesting that the eIF4A binding domains may serve a regulatory role, with the C-terminal binding site having negative effects. eIF4E binding to eIF4G601-1196 induced a conformational change, significantly increasing the binding affinity to BTE. A comparison of the binding of eIF4G deletion mutants with BTEs containing mutations showed a general correlation between binding affinity and ability to facilitate translation. In summary, these results reveal a new role for the helicase complex in 3' cap-independent translation element-mediated translation and show that the functional core domain of eIF4G plus an adjacent probable RNA-binding domain mediate translation initiation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities., Competing Interests: The authors have declared that no competing interests exist.

IGF-2 mRNA binding protein 3 (IGF2BP3, IMP-3) is a well-known post-transcriptional regulatory factor of gene expression, mainly involved in embryonic development and oncogenesis. We have previously demonstrated that a subset of IMP-3 targets, such as the mRNAs of cyclins D1, D3 and G1, are positively regulated by IMP-3, and that this regulation depends on nuclear localization of IMP-3. In the present study, we show that as a first step following a knock-down of IMP-3, the protein levels of the cyclins rapidly decrease, while their mRNAs remain stable and associated with the polyribosomes, though not translated. We have elucidated the molecular mechanisms of this regulation, demonstrating that IMP-3 and its protein partners ILF3/NF90 and PTBP1 bind to the 3'UTRs of the cyclin mRNAs and protect them from the translational repression induced by miRNA-dependent recruitment of AGO2/GW182 complex in human cancer cells.

DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5' untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F-poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5' UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly., Competing Interests: The authors declare no conflict of interest.

The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK-eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK-eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival., Competing Interests: L.C.C. is a member of the Board of Directors of, and holds equity in Agios Pharmaceuticals, a company that is developing drugs that target cancer metabolism. L.C.C. is also a founder of and holds equity in Petra Pharmaceuticals. The data presented in this manuscript are unrelated to research at Agios Pharmaceuticals or Petra Pharmaceuticals. The phosphorylation of eIF4B and related methods of use reported in this study are covered in the following published patent application: WO 2015073509 A2.

Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis., (© 2016 Signer et al.; Published by Cold Spring Harbor Laboratory Press.)

Budding yeast Tsr1 is a ribosome biogenesis factor with sequence similarity to GTPases, which is essential for cytoplasmic steps in 40S subunit maturation. Here we present the crystal structure of Tsr1 at 3.6 Å. Tsr1 has a similar domain architecture to translational GTPases such as EF-Tu and the selenocysteine incorporation factor SelB. However, active site residues required for GTP binding and hydrolysis are absent, explaining the lack of enzymatic activity in previous analyses. Modelling of Tsr1 into cryo-electron microscopy maps of pre-40S particles shows that a highly acidic surface of Tsr1 is presented on the outside of pre-40S particles, potentially preventing premature binding to 60S subunits. Late pre-40S maturation also requires the GTPase eIF5B and the ATPase Rio1. The location of Tsr1 is predicted to block binding by both factors, strongly indicating that removal of Tsr1 is an essential step during cytoplasmic maturation of 40S ribosomal subunits.

The mitochondrial genome almost exclusively encodes a handful of transmembrane constituents of the oxidative phosphorylation (OXPHOS) system. Coordinated expression of these genes ensures the correct stoichiometry of the system's components. Translation initiation in mitochondria is assisted by two general initiation factors mIF2 and mIF3, orthologues of which in bacteria are indispensible for protein synthesis and viability. mIF3 was thought to be absent in Saccharomyces cerevisiae until we recently identified mitochondrial protein Aim23 as the missing orthologue. Here we show that, surprisingly, loss of mIF3/Aim23 in S. cerevisiae does not indiscriminately abrogate mitochondrial translation but rather causes an imbalance in protein production: the rate of synthesis of the Atp9 subunit of F1F0 ATP synthase (complex V) is increased, while expression of Cox1, Cox2 and Cox3 subunits of cytochrome c oxidase (complex IV) is repressed. Our results provide one more example of deviation of mitochondrial translation from its bacterial origins.

Regulated mRNA translation is vital for germ cells to produce new proteins in the spatial and temporal patterns that drive gamete development. Translational control involves the de-repression of stored mRNAs and their recruitment by eukaryotic initiation factors (eIFs) to ribosomes. C. elegans expresses five eIF4Es (IFE-1-IFE-5); several have been shown to selectively recruit unique pools of mRNA. Individual IFE knockouts yield unique phenotypes due to inefficient translation of certain mRNAs. Here, we identified mRNAs preferentially translated through the germline-specific eIF4E isoform IFE-1. Differential polysome microarray analysis identified 77 mRNAs recruited by IFE-1. Among the IFE-1-dependent mRNAs are several required for late germ cell differentiation and maturation. Polysome association of gld-1, vab-1, vpr-1, rab-7 and rnp-3 mRNAs relies on IFE-1. Live animal imaging showed IFE-1-dependent selectivity in spatial and temporal translation of germline mRNAs. Altered MAPK activation in oocytes suggests dual roles for IFE-1, both promoting and suppressing oocyte maturation at different stages. This single eIF4E isoform exerts positive, selective translational control during germ cell differentiation., (© 2015. Published by The Company of Biologists Ltd.)

Dysregulated expression of translation initiation factors has been associated with carcinogenesis, but underlying mechanisms remains to be fully understood. Here we show that eIF4H (eukaryotic translation initiation factor 4H), an activator of the RNA helicase eIF4A, is overexpressed in lung carcinomas and predictive of response to chemotherapy. In lung cancer cells, depletion of eIF4H enhances sensitization to chemotherapy, decreases cell migration and inhibits tumor growth in vivo, in association with reduced translation of mRNA encoding cell-proliferation (c-Myc, cyclin D1) angiogenic (FGF-2) and anti-apoptotic factors (CIAP-1, BCL-xL). Conversely, each isoform of eIF4H acts as an oncogene in NIH3T3 cells by stimulating transformation, invasion, tumor growth and resistance to drug-induced apoptosis together with increased translation of IRES-containing or structured 5'UTR mRNAs. These results demonstrate that eIF4H plays a crucial role in translational control and can promote cellular transformation by preferentially regulating the translation of potent growth and survival factor mRNAs, indicating that eIF4H is a promising new molecular target for cancer therapy.

We earlier reported 'PeIF5B' as a novel factor from Pisum sativum that has sequence similarity to eIF5B (S. Rasheedi, S. Ghosh, M. Suragani et al., P. sativum contains a factor with strong homology to eIF5B, Gene 399 (2007) 144-151). The main aim of the present study was to perform functional characterization of PeIF5B as an eIF5B homologue from plant system. PeIF5B shows binding to Met - tRNA(f)(Met), hydrolyses GTP and interacts with ribosomes. In vivo growth complementation analysis shows that PeIF5B partially complements its yeast homologue. Interestingly, PeIF5B mainly localizes in the nucleus as confirmed by nuclear localization signal (NLS) prediction, confocal imaging and immunoblots of cellular fractions. Similar to the yeast eIF5B but unlike the human orthologue, PeIF5B is an intron-less gene. This study highlights PeIF5B's role as a functional eIF5B homologue possibly participating in nuclear translation in plant system., (Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Here we discuss the function of eukaryotic initiation factor 6 (eIF6; Tif6 in yeast). eIF6 binds 60S ribosomal subunits and blocks their joining to 40S. In this context, we propose that eIF6 impedes unproductive 80S formation, namely, the formation of 80S subunits without mRNA. Genetic evidence shows that eIF6 has a dual function: in yeast and mammals, nucleolar eIF6 is necessary for the biogenesis of 60S subunits. In mammals, cytoplasmic eIF6 is required for insulin and growth factor-stimulated translation. In contrast to other translation factors, eIF6 activity is not under mTOR control. The physiological significance of eIF6 impacts on cancer and on inherited Shwachman-Bodian-Diamond syndrome. eIF6 is overexpressed in specific human tumors. In a murine model of lymphomagenesis, eIF6 depletion leads to a striking increase of survival, without adverse effects. Shwachman-Bodian-Diamond syndrome is caused by loss of function of SBDS protein. In yeast, point mutations of Tif6, the yeast homolog of eIF6, rescue the quasi-lethal effect due to the loss of the SBDS homolog, Sdo1. We propose that eIF6 is a node regulator of ribosomal function and predict that prioritizing its pharmacological targeting will be of benefit in cancer and Shwachman-Bodian-Diamond syndrome. This article is part of a Special Issue entitled: Translation and Cancer., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Protein synthesis is a major energy-consuming process, which is rapidly repressed upon energy stress by AMPK. How energy deficiency affects translation of mRNAs that cope with the stress response is poorly understood. We found that mitochondrial genes remain translationally active upon energy deprivation. Surprisingly, inhibition of translation is partially retained in AMPKα1/AMPKα2 knockout cells. Mitochondrial mRNAs are enriched with TISU, a translation initiator of short 5' UTR, which confers resistance specifically to energy stress. Purified 48S preinitiation complex is sufficient for initiation via TISU AUG, when preceded by a short 5' UTR. eIF1 stimulates TISU but inhibits non-TISU-directed initiation. Remarkably, eIF4GI shares this activity and also interacts with eIF1. Furthermore, eIF4F is released upon 48S formation on TISU. These findings describe a specialized translation tolerance mechanism enabling continuous translation of TISU genes under energy stress and reveal that a key step in start codon selection of short 5' UTR is eIF4F release., (Copyright © 2015 Elsevier Inc. All rights reserved.)

During cap-dependent eukaryotic translation initiation, ribosomes scan messenger RNA from the 5' end to the first AUG start codon with favourable sequence context. For many mRNAs this AUG belongs to a short upstream open reading frame (uORF), and translation of the main downstream ORF requires re-initiation, an incompletely understood process. Re-initiation is thought to involve the same factors as standard initiation. It is unknown whether any factors specifically affect translation re-initiation without affecting standard cap-dependent translation. Here we uncover the non-canonical initiation factors density regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT-1; also called MCTS1 in humans) as the first selective regulators of eukaryotic re-initiation. mRNAs containing upstream ORFs with strong Kozak sequences selectively require DENR-MCT-1 for their proper translation, yielding a novel class of mRNAs that can be co-regulated and that is enriched for regulatory proteins such as oncogenic kinases. Collectively, our data reveal that cells have a previously unappreciated translational control system with a key role in supporting proliferation and tissue growth.

Oocyte quality is a well-established determinant of embryonic fate. However, the molecular participants and biological markers that affect and may predict adequate embryonic development are largely elusive. Our aim was to identify the components of the oocyte molecular machinery that part take in the production of a healthy embryo. For this purpose, we used an animal model, generated by us previously, the oocytes of which do not express Cx43 (Cx43(del/del)). In these mice, oogenesis appears normal, fertilisation does occur, early embryonic development is successful but implantation fails. We used magnetic resonance imaging analysis combined with histological examination to characterise the embryonic developmental incompetence. Reciprocal embryo transfer confirmed that the blastocyst evolved from the Cx43(del/del) oocyte is responsible for the implantation disorder. In order to unveil the genes, the impaired expression of which brings about the development of defective embryos, we carried out a genomic screening of both the oocytes and the resulting blastocysts. This microarray analysis revealed a low expression of Egr1, Rpl21 and Eif4a1 in Cx43(del/del) oocytes and downregulation of Rpl15 and Eif4g2 in the resulting blastocysts. We propose that global deficiencies in genes related to the expression of ribosomal proteins and translation initiation factors in apparently normal oocytes bring about accumulation of defects, which significantly compromise their developmental capacity. The blastocysts resulting from such oocytes, which grow within a confined space until implantation, may be unable to generate enough biological mass to allow their expansion. This information could be implicated to diagnosis and treatment of infertility, particularly to IVF., (© 2014 Society for Reproduction and Fertility.)

Hypoxia is considered to be a contributor to the pathology associated with administration of anthrax lethal toxin (LT). However, we report here that serum lactate levels in LT-treated mice are reduced, a finding inconsistent with the anaerobic metabolism expected to occur during hypoxia. Reduced lactate levels are also observed in the culture supernatants of LT-treated cells. LT inhibits the accumulation of hypoxia-inducible factor (HIF)-1α, a subunit of HIF-1, the master regulator directing cellular responses to hypoxia. The toxin has no effect on the transcription or protein turnover of HIF-1α, but instead it acts to inhibit HIF-1α translation. LT treatment diminishes phosphorylation of eIF4B, eIF4E, and rpS6, critical components of the intracellular machinery required for HIF-1α translation. Moreover, blockade of MKK1/2-ERK1/2, but not p38 or JNK signaling, lowers HIF-1α protein levels in both normoxic and hypoxic conditions, consistent with a role for MKK1 and MKK2 as the major targets of LT responsible for the inhibition of HIF-1α translation. The physiological importance of the LT-induced translation blockade is demonstrated by the finding that LT treatment decreases the survival of hepatocyte cell lines grown in hypoxic conditions, an effect that is overcome by preinduction of HIF-1α. Taken together, these data support a role for LT in dysregulating HIF-1α and thereby disrupting homeostatic responses to hypoxia, an environmental characteristic of certain tissues at baseline and/or during disseminated infection with Bacillus anthracis.

Hyperconnectivity of neuronal circuits due to increased synaptic protein synthesis is thought to cause autism spectrum disorders (ASDs). The mammalian target of rapamycin (mTOR) is strongly implicated in ASDs by means of upstream signalling; however, downstream regulatory mechanisms are ill-defined. Here we show that knockout of the eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2)-an eIF4E repressor downstream of mTOR-or eIF4E overexpression leads to increased translation of neuroligins, which are postsynaptic proteins that are causally linked to ASDs. Mice that have the gene encoding 4E-BP2 (Eif4ebp2) knocked out exhibit an increased ratio of excitatory to inhibitory synaptic inputs and autistic-like behaviours (that is, social interaction deficits, altered communication and repetitive/stereotyped behaviours). Pharmacological inhibition of eIF4E activity or normalization of neuroligin 1, but not neuroligin 2, protein levels restores the normal excitation/inhibition ratio and rectifies the social behaviour deficits. Thus, translational control by eIF4E regulates the synthesis of neuroligins, maintaining the excitation-to-inhibition balance, and its dysregulation engenders ASD-like phenotypes.

Eukaryotic translation initiation factor eIF4B is necessary for ribosomal scanning through structured mRNA leaders. In higher eukaryotes, eIF4B serves as a downstream effector of several signaling pathways. In response to mitogenic stimuli, eIF4B undergoes multiple phosphorylations which are thought to regulate its activity. Recently, Ser422 was identified as a predominant site for human eIF4B phosphorylation via several signaling pathways, and phosphomimetic amino acid substitutions S422D or S422E were shown to activate eIF4B in living cells. However, stimulatory role of these modifications has never been analyzed directly. Here, using both mammalian reconstituted translation initiation assay and complete cell-free translation system, we perform a comparison of recombinant eIF4B derivatives with the wild type recombinant protein, and do not find any difference in their activities. On the contrary, native eIF4B purified from HeLa cells reveals significantly higher activity in both assays. Thus, the effects of S422D and S422E substitutions on eIF4B activity in living cells observed previously either require some other protein modification(s), or may only be manifested in an intact cell. Our study raises the question on whether the phosphorylation of Ser422 is sufficient for eIF4B activation observed upon mitogenic stimulation., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Cephalopods have relatively high rates of protein synthesis compared to rates of protein degradation, along with minimal carbohydrate and lipid reserves. During food deprivation on board protein is catabolized as a metabolic fuel. The aim of the current study was to assess whether biochemical indices of protein synthesis and proteolytic mechanisms were altered in cuttlefish, Sepia officinalis, starved for 7 days. In mantle muscle, food deprivation is associated with a decrease in protein synthesis, as indicated by a decrease in the total RNA level and dephosphorylation of key signaling molecules, such as the eukaryote binding protein, 4E-BP1 (regulator of translation) and Akt. The ubiquitination-proteasome system (UPS) is activated as shown by an increase in the levels of proteasome β-subunit mRNA, polyubiquitinated protein, and polyubiquitin mRNA. As well, cathepsin activity levels are increased, suggesting increased proteolysis through the lysosomal pathway. Together, these mechanisms could supply amino acids as metabolic fuels. In gill, the situation is quite different. It appears that during the first stages of starvation, both protein synthesis and protein degradation are enhanced in gill. This is based upon increased phosphorylation of 4E-BP1 and enhanced levels of UPS indicators, especially 20S proteasome activity and polyubiquitin mRNA. It is proposed that an increased protein turnover is related to gill remodeling perhaps to retain essential hemolymph-borne compounds.

LEPA is one of the most conserved translation factors and is found from bacteria to higher plants. However, the physiological function of the chloroplast LEPA homolog in higher plants remains unknown. Herein, we demonstrate the physiological role of cpLEPA in enabling efficient photosynthesis in higher plants. The cplepa-1 mutant displays slightly high chlorophyll fluorescence and pale green phenotypes under normal growth conditions. The growth of the cplepa-1 mutant is reduced when grown on soil, and greater reduction is observed under intense light illumination. Photosynthetic activity is impaired in the cplepa-1 mutants, which is reflected in the decreased steady-state levels of chloroplast proteins. In vivo protein labeling experiments explained the decrease in the steady-state levels of chloroplast proteins. An abnormal association of the chloroplast-encoded mRNAs with ribosomes suggests that the protein synthesis deficiencies in cplepa-1 are due to defects in translation initiation in the chloroplasts. The cpLEPA protein appears to be an essential translation factor that promotes the efficiency of chloroplast protein synthesis.

Open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus (KSHV) causes sustained activation of p90 ribosomal S6 kinase (RSK), which is crucial for KSHV lytic replication, but the exact functional roles remain to be determined. To characterize the biological consequence of persistent RSK activation by ORF45, we screened known cellular substrates of RSK. We found that ORF45 induced phosphorylation of eukaryotic translation initiation factor 4B (eIF4B), increased its assembly into translation initiation complex, and subsequently facilitated protein translation. The ORF45/RSK-mediated eIF4B phosphorylation was distinguishable from that caused by the canonical AKT/mammalian target of rapamycin/ribosomal S6 kinase and MEK/ERK/RSK pathways because it was resistant to both rapamycin (an mammalian target of rapamycin inhibitor) and U1026 (an MEK inhibitor). The rapamycin and U1026 doubly insensitive eIF4B phosphorylation was induced during KSHV reactivation but was abolished if either ORF45 or RSK1/2 were ablated by siRNA, a pattern that is correlated with reduced lytic gene expression as we observed previously. Ectopic expression of eIF4B but not its phosphorylation-deficient mutant form increased KSHV lytic gene expression and production of progeny viruses. Together, these results indicated that ORF45/RSK axis-induced eIF4B phosphorylation is involved in translational regulation and is required for optimal KSHV lytic replication.

Translational control is one of primary regulation mechanisms of gene expression. Eukaryotic translational control mainly occurs at the initiation step, the speed-limiting step, which involves more than ten translation initiation factors [eIFs (eukaryotic initiation factors)]. Changing the level or function of these eIFs results in abnormal translation of specific mRNAs and consequently abnormal growth of cells that leads to human diseases, including cancer. Accumulating evidence from recent studies showed that the expression of many eIFs was associated with malignant transformation, cancer prognosis, as well as gene expression regulation. In the present paper, we perform a critical review of recent advances in understanding the role and mechanism of eIF action in translational control and cancer as well as the possibility of targeting eIFs for therapeutic development.

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3'-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

Alzheimer's disease (AD) is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta), a cleavage product of amyloid precursor protein (APP). Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective.