eIF4B mRNA Translation Contributes to Cleavage Dynamics in Early Sea Urchin Embryos.

During the first steps of sea urchin development, fertilization elicits a marked increase in protein synthesis essential for subsequent cell divisions. While the translation of mitotic cyclin mRNAs is crucial, we hypothesized that additional mRNAs must be translated to finely regulate the onset into mitosis. One of the maternal mRNAs recruited onto active polysomes at this stage codes for the initiation factor eIF4B. Here, we show that the sea urchin eIF4B orthologs present the four specific domains essential for eIF4B function and that Paracentrotus lividus eIF4B copurifies with eIF4E in a heterologous system. In addition, we investigated the role of eIF4B mRNA de novo translation during the two first embryonic divisions of two species, P. lividus and Sphaerechinus granularis. Our results show that injection of a morpholino directed against eIF4B mRNA results in a downregulation of translational activity and delays cell division in these two echinoids. Conversely, injection of an mRNA encoding for P. lividus eIF4B stimulates translation and significantly accelerates cleavage rates. Taken together, our findings suggest that eIF4B mRNA de novo translation participates in a conserved regulatory loop that contributes to orchestrating protein synthesis and modulates cell division rhythm during early sea urchin development.

In vivo analysis of protein translation activity in sea urchin eggs and embryos.

Protein synthesis is a major regulatory step of gene expression in different physiological processes including development. Translation of proteins in sea urchin is stimulated upon fertilization and is necessary for cell cycle progression and development. Translational control is exerted through multifactorial mechanisms, including mRNA recruitment into polysomes and increased rates of translational activity. In this chapter, we review the methods used in sea urchin eggs and embryos to analyze translation activity in vivo both from perspectives of the proteins and of the mRNAs. First, we describe methods to quantify or visualize newly synthesized proteins with radioactive and non-radioactive labeling techniques. Next we present the polysome isolation and profiling on sucrose gradients, allowing the identification of translated mRNAs. Finally, we outline a procedure to follow the translation of a reporter luciferase protein from an mRNA microinjected into the egg.

Translational Control of Canonical and Non-Canonical Translation Initiation Factors at the Sea Urchin Egg to Embryo Transition.

Sea urchin early development is a powerful model to study translational regulation under physiological conditions. Fertilization triggers an activation of the translation machinery responsible for the increase of protein synthesis necessary for the completion of the first embryonic cell cycles. The cap-binding protein eIF4E, the helicase eIF4A and the large scaffolding protein eIF4G are assembled upon fertilization to form an initiation complex on mRNAs involved in cap-dependent translation initiation. The presence of these proteins in unfertilized and fertilized eggs has already been demonstrated, however data concerning the translational status of translation factors are still scarce. Using polysome fractionation, we analyzed the impact of fertilization on the recruitment of mRNAs encoding initiation factors. Strikingly, whereas the mRNAs coding eIF4E, eIF4A, and eIF4G were not recruited into polysomes at 1 h post-fertilization, mRNAs for eIF4B and for non-canonical initiation factors such as DAP5, eIF4E2, eIF4E3, or hnRNP Q, are recruited and are differentially sensitive to the activation state of the mechanistic target of rapamycin (mTOR) pathway. We discuss our results suggesting alternative translation initiation in the context of the early development of sea urchins.

Translatome analysis at the egg-to-embryo transition in sea urchin.

Early embryogenesis relies on the translational regulation of maternally stored mRNAs. In sea urchin, fertilization triggers a dramatic rise in translation activity, necessary for the onset of cell division. Here, the full spectrum of the mRNAs translated upon fertilization was investigated by polysome profiling and sequencing. The translatome of the early sea urchin embryo gave a complete picture of the polysomal recruitment dynamics following fertilization. Our results indicate that only a subset of maternal mRNAs were selectively recruited onto polysomes, with over-represented functional categories in the translated set. The increase in translation upon fertilization depends on the formation of translation initiation complexes following mTOR pathway activation. Surprisingly, mTOR pathway inhibition differentially affected polysomal recruitment of the newly translated mRNAs, which thus appeared either mTOR-dependent or mTOR-independent. Therefore, our data argue for an alternative to the classical cap-dependent model of translation in early development. The identification of the mRNAs translated following fertilization helped assign translational activation events to specific mRNAs. This translatome is the first step to a comprehensive analysis of the molecular mechanisms governing translation upon fertilization and the translational regulatory networks that control the egg-to-embryo transition as well as the early steps of embryogenesis.

Analysis of translation using polysome profiling.

During the past decade, there has been growing interest in the role of translational regulation of gene expression in many organisms. Polysome profiling has been developed to infer the translational status of a specific mRNA species or to analyze the translatome, i.e. the subset of mRNAs actively translated in a cell. Polysome profiling is especially suitable for emergent model organisms for which genomic data are limited. In this paper, we describe an optimized protocol for the purification of sea urchin polysomes and highlight the critical steps involved in polysome purification. We applied this protocol to obtain experimental results on translational regulation of mRNAs following fertilization. Our protocol should prove useful for integrating the study of the role of translational regulation in gene regulatory networks in any biological model. In addition, we demonstrate how to carry out high-throughput processing of polysome gradient fractions, for the simultaneous screening of multiple biological conditions and large-scale preparation of samples for next-generation sequencing.

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos.

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism.

mRNA-selective translation induced by FSH in primary Sertoli cells.

FSH is a key hormonal regulator of Sertoli cell secretory activity, required to optimize sperm production. To fulfil its biological function, FSH binds a G protein-coupled receptor, the FSH-R. The FSH-R-transduced signaling network ultimately leads to the transcription or down-regulation of numerous genes. In addition, recent evidence has suggested that FSH might also regulate protein translation. However, this point has never been demonstrated conclusively yet. Here we have addressed this issue in primary rat Sertoli cells endogenously expressing physiological levels of FSH-R. We observed that, within 90 min of stimulation, FSH not only enhanced overall protein synthesis in a mammalian target of rapamycin-dependent manner but also increased the recruitment of mRNA to polysomes. m(7)GTP pull-down experiments revealed the functional recruitment of mammalian target of rapamycin and p70 S6 kinase to the 5'cap, further supported by the enhanced phosphorylation of one of p70 S6 kinase targets, the eukaryotic initiation factor 4B. Importantly, the scaffolding eukaryotic initiation factor 4G was also recruited, whereas eukaryotic initiation factor 4E-binding protein, the eukaryotic initiation factor 4E generic inhibitor, appeared to play a minor role in translational regulations induced by FSH, in contrast to what is generally observed in response to anabolic factors. This particular regulation of the translational machinery by FSH stimulation might support mRNA-selective translation, as shown here by quantitative RT-PCR amplification of the c-fos and vascular endothelial growth factor mRNA but not of all FSH target mRNA, in polysomal fractions. These findings add a new level of complexity to FSH biological roles in its natural target cells, which has been underappreciated so far.

A variant mimicking hyperphosphorylated 4E-BP inhibits protein synthesis in a sea urchin cell-free, cap-dependent translation system.

BACKGROUND: 4E-BP is a translational inhibitor that binds to eIF4E to repress cap-dependent translation initiation. This critical protein:protein interaction is regulated by the phosphorylation of 4E-BP. Hypophosphorylated 4E-BP binds to eIF4E and inhibits cap-dependent translation, whereas hyperphosphorylated forms do not. While three 4E-BP proteins exist in mammals, only one gene encoding for 4E-BP is present in the sea urchin genome. The protein product has a highly conserved core domain containing the eIF4E-binding domain motif (YxxxxLPhi) and four of the regulatory phosphorylation sites. METHODOLOGY/PRINCIPAL FINDINGS: Using a sea urchin cell-free cap-dependent translation system prepared from fertilized eggs, we provide the first direct evidence that the sea urchin 4E-BP inhibits cap-dependent translation. We show here that a sea urchin 4E-BP variant, mimicking phosphorylation on four core residues required to abrogate binding to eIF4E, surprisingly maintains physical association to eIF4E and inhibits protein synthesis. CONCLUSIONS/SIGNIFICANCE: Here, we examine the involvement of the evolutionarily conserved core domain and phosphorylation sites of sea urchin 4E-BP in the regulation of eIF4E-binding. These studies primarily demonstrate the conserved activity of the 4E-BP translational repressor and the importance of the eIF4E-binding domain in sea urchin. We also show that a variant mimicking hyperphosphorylation of the four regulatory phosphorylation sites common to sea urchin and human 4E-BP is not sufficient for release from eIF4E and translation promotion. Therefore, our results suggest that there are additional mechanisms to that of phosphorylation at the four critical sites of 4E-BP that are required to disrupt binding to eIF4E.

Inhibition of translation and modification of translation factors during apoptosis induced by the DNA-damaging agent MMS in sea urchin embryos.

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.

Cellular coexistence of two high molecular subsets of eEF1B complex.

The elongation factor eEF1B involved in protein translation was found to contain two isoforms of the eEF1Bdelta subunit in sea urchin eggs. The eEF1Bdelta2 isoform differs from eEF1Bdelta1 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 eEF1Bdelta2 antibodies. Quantification of the proteins in immunoprecipitates and on immunoblots demonstrates that eEF1Bdelta1 and eEF1Bdelta2 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.

eEF1B: At the dawn of the 21st century.

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.

Embryonic-stage-dependent changes in the level of eIF4E-binding proteins during early development of sea urchin embryos.

The eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) inhibit translation initiation by binding eIF4E and preventing recruitment of the translation machinery to mRNA. We have previously shown that fertilization of sea urchin eggs triggers eIF4E-4E-BP complex dissociation and 4E-BP degradation. Here, we show that microinjection of eIF4E-binding motif peptide into unfertilized eggs delays the onset of the first mitosis triggered by fertilization, demonstrating that dissociation of the eIF4E-4E-BP complex is functionally important for the first mitotic division in sea urchin embryos. We also show by gel filtration analyses that eIF4E is present in unfertilized eggs as an 80 kDa molecular mass complex containing 4E-BP and a new 4E-BP of 40 kDa. Fertilization triggers the dissociation of eIF4E from these two 4E-BPs and triggers the rapid recruitment of eIF4E into a high-molecular-mass complex. Release of eIF4E from the two 4E-BPs is correlated with a decrease in the total level of both 4E-BPs following fertilization. Abundance of the two 4E-BPs has been monitored during embryonic development. The level of the two proteins remains very low during the rapid cleavage stage of early development and increases 8 hours after fertilization. These results demonstrate that these two 4E-BPs are down- and upregulated during the embryonic development of sea urchins. Consequently, these data suggest that eIF4E availability to other partners represents an important determinant of the early development of sea urchin embryos.

Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos.

In sea urchins, fertilization triggers a rapid rise in protein synthesis necessary for activation of CDK1/cyclin B, the universal cell cycle regulator. It has been shown that FRAP/mTOR is required for eIF4E release from the translational repressor 4E-BP, a process that occurs upstream of de novo cyclin B synthesis. Here, we investigate whether PI 3-kinase acts independently or upstream from FRAP/mTOR in the signal transduction pathway that links fertilization to the activation of the CDK1/cyclin B complex in sea urchin egg. We found that wortmannin, a potent inhibitor of PI 3-kinase, partially inhibited the global increase in protein synthesis triggered by fertilization. Furthermore, wortmannin treatment induced partial inhibition of cyclin B translation triggered by fertilization, in correlation with an intermediate effect of the drug on 4E-BP degradation and on the dissociation of the 4E-BP/eIF4E complex induced by fertilization. Our results presented here suggest that PI 3-kinase activity is required for completion of mitotic divisions of the sea urchin embryo. Incubation of eggs with wortmannin or microinjection of wortmannin or LY 294002 affects drastically mitotic divisions induced by fertilization. In addition, we found that wortmannin treatment inhibits dephosphorylation of the tyrosine inhibitory site of CDK1. Taken together, these data suggest that PI 3-kinase acts upstream of at least two independent targets that function in the CDK1/cyclin B activation triggered by fertilization of sea urchin oocytes. We discuss the significance of these results concerning the cascade of reactions that impinge upon the activation of the CDK1/cyclin B complex that follows sea urchin oocyte fertilization.

Pesticide Roundup provokes cell division dysfunction at the level of CDK1/cyclin B activation.

To assess human health risk from environmental chemicals, we have studied the effect on cell cycle regulation of the widely used glyphosate-containing pesticide Roundup. As a model system we have used sea urchin embryonic first divisions following fertilization, which are appropriate for the study of universal cell cycle regulation without interference with transcription. We show that 0.8% Roundup (containing 8 mM glyphosate) induces a delay in the kinetic of the first cell cleavage of sea urchin embryos. The delay is dependent on the concentration of Roundup. The delay in the cell cycle could be induced using increasing glyphosate concentrations (1-10 mM) in the presence of a subthreshold concentration of Roundup 0.2%, while glyphosate alone was ineffective, thus indicating synergy between glyphosate and Roundup formulation products. The effect of Roundup was not lethal and involved a delay in entry into M-phase of the cell cycle, as judged cytologically. Since CDK1/cyclin B regulates universally the M-phase of the cell cycle, we analyzed CDK1/cyclin B activation during the first division of early development. Roundup delayed the activation of CDK1/cyclin B in vivo. Roundup inhibited also the global protein synthetic rate without preventing the accumulation of cyclin B. In summary, Roundup affects cell cycle regulation by delaying activation of the CDK1/cyclin B complex, by synergic effect of glyphosate and formulation products. Considering the universality among species of the CDK1/cyclin B regulator, our results question the safety of glyphosate and Roundup on human health.