Patrick Cormier

Analysis of translation using polysome profiling

Abstract 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.

Modelization of the regulation of protein synthesis following fertilization in sea urchin shows requirement of two processes: a destabilization of eIF4E:4E-BP complex and a great stimulation of the 4E-BP-degradation mechanism, both rapamycin-sensitive

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.

Translation regulator ballet in meiotic spindle

The fine spatial and temporal resolution of translation control can have a rapid and subtle effect on the microenvironment of the cell in comparison with transcriptional regulation. Mammalian oocyte represents a relevant model that allows addressing the spatial control of translation during meiotic maturation. The fully-grown mammalian oocyte is transcriptionally quiescent and stored maternal RNAs are used for the completion of meiosis and early embryo development. It was shown three decades ago that during resumption of meiosis, protein synthesis is not necessary for the nuclear envelope breakdown (NEBD) but active protein synthesis is required for the correct formation of the meiotic spindle and progression to metaphase II (1). Therefore mammalian oocyte represents a suited system to address translation control in relation with the cell cycle in general and with the spindle formation in particular. In this issue of the Cell Cycle the study by Jansova et al. gains new insight into the role of the eukaryotic initiation factor 4E-Binding Protein 1 (4E-BP1) in the meiotic spindle formation in mouse oocyte (2).

Folate-conjugated stealth archaeosomes for the targeted delivery of novel antitumoral peptides

In this work, novel archaeosomes based on Egg-PC and a mixture of PEGylated archaeal tetraether lipids were investigated as nanocarriers for in vitro delivery of an original anticancer peptide. With the aim to develop site-specific drug targeting, a tetraether equipped with a folate ligand at the PEG5000 terminal end (FA-PEG5000-tetraether) was synthesized in order to bind to folate receptors (over)expressed on the tumor cell surfaces. The original peptide A1 and its inactive analogue A1Yala (17 amino acids) were encapsulated into Egg-PC vesicles incorporating FA-PEG5000-tetraether and/or PEG2000-tetraether lipids in order to evaluate the in vitro anticancer activity of A1-loaded archaeosomes. Results showed a particular behaviour when A1 was encapsulated into the folate-equipped archaeosomes particularly during the first hour of incubation.

Model of the delayed translation of cyclin B maternal mRNA after sea urchin fertilization

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 (ka) of 0.024 sec−1, for the activation of XXmRNAcyclinB, and a critical time interval (t2) 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.

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

Abstract 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.

Toward Multiscale Modeling of Molecular and Biochemical Events Occurring at Fertilization Time in Sea Urchins

We review here previous theoretical and experimental works, which aim to model major events that occur at the time of fertilization in the sea urchin. We discuss works that perform experiments and develop hypotheses that link different scales of biological systems such as the intracellular Ca2+ concentration oscillations and the swimming behavior of sperm, the Ca2+ wave propagation and the fertilization membrane elevation of the egg, and the mRNA translational activation and the completion of the first mitotic division of the early embryo. The aim of this review is on one hand, to highlight the value of systems biology for understanding the mechanisms associated with fertilization and early embryonic development in sea urchins. On the other hand, this review attempts to illustrate, for mathematicians and bioinformaticians, the potential that represent these molecular and cellular events for modeling clear physiological processes.