SUMMARY Sea urchin eggs exhibit a cap‐dependent increase in protein synthesis within minutes after fertilization. This rise in protein synthesis occurs at a constant rate for a great number of proteins translated from the different available mRNAs. Surprisingly, we found that cyclin B, a major cell‐cycle regulator, follows a synthesis pattern that is distinct from the global protein population, so we developed a mathematical model to analyze this dissimilarity in biosynthesis kinetic patterns. The model includes two pathways for cyclin B mRNA entry into the translational machinery: one from immediately available mRNA (mRNAcyclinB) and one from mRNA activated solely after fertilization (XXmRNAcyclinB). Two coefficients, α and β, were added to fit the measured scales of global protein and cyclin B synthesis, respectively. The model was simplified to identify the synthesis parameters and to allow its simulation. The calculated parameters for activation of the specific cyclin B synthesis pathway after fertilization included a kinetic constant (k a ) of 0.024 sec −1 , for the activation of XXmRNAcyclinB, and a critical time interval (t 2 ) of 42 min. The proportion of XXmRNAcyclinB form was also calculated to be largely dominant over the mRNAcyclinB form. Regulation of cyclin B biosynthesis is an example of a select protein whose translation is controlled by pathways that are distinct from housekeeping proteins, even though both involve the same cap‐dependent initiation pathway. Therefore, this model should help provide insight to the signaling utilized for the biosynthesis of cyclin B and other select proteins. Mol. Reprod. Dev. 83: 1070–1082, 2016. © 2016 Wiley Periodicals, Inc .

Using sea urchin embryos, we demonstrate that the MEK/MAPK/ERK cascade is essential for the proper progression of the cell cycle. Activation of a limited fraction of MAPK/ERK is required between S-phase and M-phase. Neither DNA replication nor CDK1 activation are impacted by the inhibition of this small active MAPK/ERK fraction. Nonetheless, the chromatin and spindle organisations are profoundly altered. Early morphological disorders induced by the absence of MAPK/ERK activation are correlated with an important inhibition of global protein synthesis and modification in the cyclin B accumulation profile. After appearance of morphological disorders, there is an increase in the level of the inhibitor of protein synthesis, 4E-BP, and, ultimately, an activation of the spindle checkpoint. Altogether, our results suggest that MAPK/ERK activity is required for the synthesis of (a) protein(s) implicated in an early step of chromatin /microtubule attachment. If this MAPK/ERK-dependent step is not achieved, the cell activates a new checkpoint mechanism, involving the reappearance of 4E-BP that maintains a low level of protein translation, thus saving cellular energy.

The International Agency for Research on Cancer (IARC) Monographs Programme identifies chemicals, drugs, mixtures, occupational exposures, lifestyles and personal habits, and physical and biological agents that cause cancer in humans and has evaluated about 1000 agents since 1971. Monographs are written by ad hoc Working Groups (WGs) of international scientific experts over a period of about 12 months ending in an eight-day meeting. The WG evaluates all of the publicly available scientific information on each substance and, through a transparent and rigorous process,1 decides on the degree to which the scientific evidence supports that substance's potential to cause or not cause cancer in humans. For Monograph 112,2 17 expert scientists evaluated the carcinogenic hazard for four insecticides and the herbicide glyphosate.3 The WG concluded that the data for glyphosate meet the criteria for classification as a probable human carcinogen . The European Food Safety Authority (EFSA) is the primary agency of the European Union for risk assessments regarding food safety. In October 2015, EFSA reported4 on their evaluation of the Renewal Assessment Report5 (RAR) for glyphosate that was prepared by the Rapporteur Member State, the German Federal Institute for Risk Assessment (BfR). EFSA concluded that ‘glyphosate is unlikely to pose a carcinogenic hazard to humans and the evidence does not support classification with regard to its carcinogenic potential’. Addendum 1 (the BfR Addendum) of the RAR5 discusses the scientific rationale for differing from the IARC WG conclusion. Serious flaws in the scientific evaluation in the RAR incorrectly characterise the potential for a carcinogenic hazard from exposure to glyphosate. Since the RAR is the basis for the European Food Safety Agency (EFSA) conclusion,4 it is critical that these shortcomings are corrected. EFSA concluded ‘that there is very limited evidence for an association between glyphosate-based formulations …

Fertilization of sea urchin eggs involves an increase in protein synthesis associated with a decrease in the amount of the translation initiation inhibitor 4E-BP. A highly simple reaction model for the regulation of protein synthesis was built and was used to simulate the physiological changes in the total 4E-BP amount observed during time after fertilization. Our study evidenced that two changes occurring at fertilization are necessary to fit with experimental data. The first change was an 8-fold increase in the dissociation parameter (koff1) of the eIF4E:4E-BP complex. The second was an important 32.5-fold activation of the degradation mechanism of the protein 4E-BP. Additionally, the changes in both processes should occur in 5 min time interval post-fertilization. To validate the model, we checked that the kinetic of the predicted 4.2-fold increase of eIF4E:eIF4G complex concentration at fertilization matched the increase of protein synthesis experimentally observed after fertilization (6.6-fold, SD = 2.3, n = 8). The minimal model was also used to simulate changes observed after fertilization in the presence of rapamycin, a FRAP/mTOR inhibitor. The model showed that the eIF4E:4E-BP complex destabilization was impacted and surprisingly, that the mechanism of 4E-BP degradation was also strongly affected, therefore suggesting that both processes are controlled by the protein kinase FRAP/mTOR.

The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.

Elongation factor 2 (eEF2) is the main regulator of peptide chain elongation in eukaryotic cells. Using sea urchin eggs and early embryos, two isoforms of eEF2 of respectively 80 and 83 kDa apparent molecular weight have been discovered. Both isoforms were identified by immunological analysis as well as mass spectrometry, and appeared to originate from a unique post-translationally modified protein. Accompanying the net increase in protein synthesis that occurs in early development, both eEF2 isoforms underwent dephosphorylation in the 15 min period following fertilization, in accordance with the active role of dephosphorylated eEF2 in regulation of protein synthesis. After initial dephosphorylation, the major 83 kDa isoform remained dephosphorylated while the 80 kDa isoform was progressively re-phosphorylated in a cell-cycle dependent fashion. In vivo inhibition of phosphorylation of the 80 kDa isoform impaired the completion of the first cell cycle of early development implicating the involvement of eEF2 phosphorylation in the exit from mitosis.

The large and rapid increase in the rate of protein synthesis following fertilization of the sea urchin egg has long been a paradigm of translational control, an important component of the regulation of gene expression in cells. This translational up-regulation is linked to physiological changes that occur upon fertilization and is necessary for entry into first cell division cycle. Accumulated knowledge on cap-dependent initiation of translation makes it suited and timely to start integrating the data into a system view of biological functions. Using a programming environment for system biology coupled with model validation (named Biocham), we have built an integrative model for cap-dependent initiation of translation. The model is described by abstract rules. It contains 51 reactions involved in 74 molecular complexes. The model proved to be coherent with existing knowledge by using queries based on computational tree logic (CTL) as well as Boolean simulations. The model could simulate the change in translation occurring at fertilization in the sea urchin model. It could also be coupled with an existing model designed for cell-cycle control. Therefore, the cap-dependent translation initiation model can be considered a first step towards the eukaryotic translation regulation network.

Translational control was investigated in sea urchin eggs and embryos in response to the DNA-damaging agent methyl methanesulfonate (MMS). We have shown in this report that exposure of sea urchin embryos to MMS induces drastic effects on protein synthesis activity, and on translation factors level, integrity and post-translational modifications. In response to the treatment of embryos by the DNA-damaging agent MMS, protein synthesis is inhibited independently of the translation inhibitor 4E-BP and in correlation with phosphorylation of the translation factor eIF2alpha subunit. Furthermore, a low molecular weight form of translation initiation factor eIF4G is detected correlatively with MMS-induced apoptosis. We propose that modifications of translation factors play an important role in protein synthesis modulation that occurs during DNA-damage induced apoptosis.

Using sea urchin early embryos as a pertinent model, chromium(III) provoked cell cycle arrest and induced apoptosis. The molecular machinery of translation initiation was investigated. Chromium provoked a time- and dose-dependent increase in the level of 4E-BP protein, the natural regulator of the cap-dependent initiation factor 4E (eIF4E). The 4E-BP increase was the result of 4E-BP stabilization and appeared functional for physiological eIF4E binding, removal of eIF4E from the initiation factor eIF4G, and almost full inhibition of cap-dependent translation in vivo. The protein 4E-BP may be involved in the biological pathway of apoptosis associated with the activation of the DNA-damaged checkpoint of the cell cycle.

mRNA translation is now recognized as a important regulatory step for gene expression in different physiological and pathophysiological processes including cell proliferation and apoptosis. B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of resting lymphocytes and defective apoptosis. The mRNA cap-binding protein eIF4E (eukaryotic Initiation Factor 4E) and its repressor 4E-BP (eIF4E Binding protein) are crucial translational regulators that have been involved in survival and apoptosis processes of cells. We have shown that the release of eIF4E from its translational repressor 4E-BP is an important event for the first mitotic division triggered by fertilization and that the degradation of 4E-BP is a new means to regulate 4E-BP function that has to be analyzed in other physiological and physiopathological processes. In this chapter, we describe recent advances illustrating the importance of eIF4E and 4E-BP in cancer processes, suggesting that these actors can be targeted for potential therapy against cancer in general and LLC in particular.

Gene expression regulation is crucial for organism survival. Each step has to be regulated, from the gene to the protein. mRNA can be stored in the cell without any direct translation. This process is used by the cell to control protein synthesis rapidly at the right place, at the right time. Protein synthesis costs a lot of energy for the cell, so that a precise control of this process is required. Translation initiation represents an important step to regulate gene expression. Many factors that can bind mRNA and recruit different partners are involved in the inhibition or stimulation of protein synthesis. Oceans contain an important diversity of organisms that are used as important models to analyse gene expression at the translational level. These are useful to study translational control in different physiological processes for instance cell cycle (meiosis during meiotic maturation of starfish oocytes, mitosis following fertilization of sea urchin eggs) or to understand nervous system mechanisms (aplysia). All these studies will help finding novel actors involved in translational control and will thus be useful to discover new targets for therapeutic treatments against human diseases.

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer.

La regulation de l'expression des genes est cruciale pour la survie des organismes, chaque etape doit etre finement regulee, depuis les genes jusqu'a la formation des proteines. Les ARNm peuvent etre stockes dans une cellule sans etre traduits automatiquement. Cela permet a la cellule de reagir rapidement pour produire les proteines necessaires au bon endroit et au bon moment en regulant l'etape de traduction. La cellule depense beaucoup d'energie pour synthetiser des proteines, il est essentiel de controler ces processus en fonction des besoins cellulaires. L'etape d'initiation de la traduction represente une etape regulatrice importante dans l'expression des genes. Elle fait intervenir de nombreux facteurs proteiques capables de se lier aux ARNm et de recruter differents partenaires pour inhiber ou stimuler la synthese proteique. Les oceans contiennent une diversite d'organismes qui constituent d'excellents modeles pour etudier les bases de l'expression des genes au niveau de la traduction. Ces organismes ont permis d'etudier des mecanismes de regulation traductionnelle dans differents processus physiologiques : cycle cellulaire (meiose lors de la maturation meiotique de l'etoile de mer, mitose en reponse a la fecondation chez l'oursin), et de mieux comprendre le fonctionnement du systeme nerveux (aplysie). Toutes ces donnees permettront de trouver de nouveaux acteurs indispensables a la regulation de la traduction et de fournir de nouvelles cibles de therapie dans la lutte contre les maladies chez l'Homme.

Cell division is an essential process for heredity, maintenance and evolution of the whole living kingdom. Sea urchin early development represents an excellent experimental model for the analysis of cell cycle checkpoint mechanisms since embryonic cells contain a functional DNA-damage checkpoint and since the whole sea urchin genome is sequenced. The DNA-damaged checkpoint is responsible for an arrest in the cell cycle when DNA is damaged or incorrectly replicated, for activation of the DNA repair mechanism, and for commitment to cell death by apoptosis in the case of failure to repair. New insights in cancer biology lead to two fundamental concepts about the very first origin of cancerogenesis. Cancers result from dysfunction of DNA-damaged checkpoints and cancers appear as a result of normal stem cell (NCS) transformation into a cancer stem cell (CSC). The second aspect suggests a new definition of "cancer", since CSC can be detected well before any clinical evidence. Since early development starts from the zygote, which is a primary stem cell, sea urchin early development allows analysis of the early steps of the cancerization process. Although sea urchins do not develop cancers, the model is alternative and complementary to stem cells which are not easy to isolate, do not divide in a short time and do not divide synchronously. In the field of toxicology and incidence on human health, the sea urchin experimental model allows assessment of cancer risk from single or combined molecules long before any epidemiologic evidence is available. Sea urchin embryos were used to test the worldwide used pesticide Roundup that contains glyphosate as the active herbicide agent; it was shown to activate the DNA-damage checkpoint of the first cell cycle of development. The model therefore allows considerable increase in risk evaluation of new products in the field of cancer and offers a tool for the discovery of molecular markers for early diagnostic in cancer biology. Prevention and early diagnosis are two decisive elements of human cancer therapy.

La traduction des ARNm est une etape regulatrice importante de l'expression des genes au cours de differents mecanismes physiologiques et physiopathologiques, incluant la proliferation cellulaire et l'apoptose. Parmi les maladies du cycle cellulaire, la leucemie lymphoide chronique B-CD5 + (LLC) est caracterisee par une perte de l'apoptose. Les proteines eIF4E (eukaryotic Initiation Factor 4E) et 4E-BPs (eIF4E Binding Proteins), qui jouent un role important dans la regulation traductionnelle, ont recemment ete impliquees dans les mecanismes de survie et d'apoptose. Nos etudes ciblant ces acteurs au cours du developpement embryonnaire precoce de l'oursin nous ont permis de souligner leur importance pour l'entree dans le cycle cellulaire en reponse a la fecondation. Dans ce modele, la degradation de 4E-BP represente un nouveau mecanisme de controle de la traduction et doit etre pris en compte dans d'autres mecanismes physiologiques et physiopathologiques. L'ensemble des donnees permet de proposer les facteurs eIF4 comme cible potentielle pour de nouvelles approches therapeutiques contre le cancer en general et contre la LLC en particulier. Cette revue met en evidence l'apport du modele biologique du developpement precoce de l'oursin dans cet axe de recherche qui associe de facon originale une equipe de recherche de biologie cellulaire et une equipe medicale specialisee dans la cancerologie de la leucemie lymphoide chronique B-CD5 + .

Sea urchin eggs and early cleavage stage embryos provide an example of regulated gene expression at the level of translation. The availability of the sea urchin genome offers the opportunity to investigate the "translational control" toolkit of this model system. The annotation of the genome reveals that most of the factors implicated in translational control are encoded by nonredundant genes in echinoderm, an advantage for future functional studies. In this paper, we focus on translation factors that have been shown or suggested to play crucial role in cell cycle and development of sea urchin embryos. Addressing the cap-binding translational control, three closely related eIF4E genes (class I, II, III) are present, whereas its repressor 4E-BP and its activator eIF4G are both encoded by one gene. Analysis of the class III eIF4E proteins in various phyla shows an echinoderm-specific amino acid substitution. Furthermore, an interaction site between eIF4G and poly(A)-binding protein is uncovered in the sea urchin eIF4G proteins and is conserved in metazoan evolution. In silico screening of the sea urchin genome has uncovered potential new regulators of eIF4E sharing the common eIF4E recognition motif. Taking together, these data provide new insights regarding the strong requirement of cap-dependent translation following fertilization. The genome analysis gives insights on the complexity of eEF1B structure and motifs of functional relevance, involved in the translational control of gene expression at the level of elongation. Finally, because deregulation of translation process can lead to diseases and tumor formation in humans, the sea urchin orthologs of human genes implicated in human diseases and signaling pathways regulating translation were also discussed.

Translational regulation of gene expression in eukaryotes can rapidly and accurately control cell activity in response to stimuli or when rapidly dividing. There is increasing evidence for a key role of the elongation step in this process. Elongation factor-1 (eEF1), which is responsible for aminoacyl-tRNA transfer on the ribosome, is comprised of two entities: a G-protein named eEF1A and a nucleotide exchange factor, eEF1B. The multifunctional nature of eEF1A, as well as its oncogenic potential, is currently the subject of a number of studies. Until recently, less work has been done on eEF1B. This review describes the macromolecular complexity of eEF1B, its multiple phosphorylation sites and numerous cellular partners, which lead us to suggest an essential role for the factor in the control of gene expression, particularly during the cell cycle.

The 4E-binding proteins (4E-BPs) regulate the cap-dependent eukaryotic initiation factor 4E (eIF4E). The level of 4E-BP protein is regulated during early development of sea urchin embryos. Fertilization leads to the rapid disappearance of the protein that reappears later in development. We show that two important cellular stresses, hypoxia and bleomycin prolonged checkpoint mobilization provoked the overexpression of the protein 4E-BP in developing sea urchin embryos. Hypoxia resulted after 1 h in a reversible gradual increase in the protein 4E-BP level. At 20 h, the protein 4E-BP had reached the level existing in the unfertilized eggs. Bleomycin used as a DNA-damaging agent for checkpoint activation, provoked cell cycle inhibition and after prolonged exposure (20 h), induced the expression of the protein 4E-BP. The effect of bleomycin on 4E-BP protein overexpression was dose-dependent between 0.4 and 1.2 mM. The role of the overexpression of the protein 4E-BP is discussed in relation with cellular stress responses.

The elongation factor eEF1B involved in protein translation was found to contain two isoforms of the eEF1Bδ subunit in sea urchin eggs. The eEF1Bδ2 isoform differs from eEF1Bδ1 by a specific insert of 26 amino acids. Both isoforms are co‐expressed in the cell and likely originate from a unique gene. The feature appears universal in metazoans as judged from in silico analysis in EST‐databanks. The eEF1B components were co‐immunoprecipitated by specific eEF1Bδ2 antibodies. Quantification of the proteins in immunoprecipitates and on immunoblots demonstrates that eEF1Bδ1 and eEF1Bδ2 proteins are present in two subsets of eEF1B complex. We discuss and propose a model for the different subsets of eEF1B complex concomitantly present in the cell.