Chassé H et al. (2018), Translatome analysis at the egg-to-embryo trans...

ECB-ART-46242

Nucleic Acids Res 2018 May 18;469:4607-4621. doi: 10.1093/nar/gky258.

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

Chassé H , Aubert J , Boulben S , Le Corguillé G , Corre E , Cormier P , Morales J .

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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.

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Species referenced: Echinodermata
Genes referenced: ccnb2 cdk1 eif4a impact LOC100887844 LOC115919910 LOC115925415 LOC583082 LOC594261

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	Figure 1. (A) Cell division kinetics in Paracentrotus lividus from two independent experiments, error bars represent standard deviation. (B) Protein synthesis activity measured by 15-min pulse-labeling in unfertilized eggs (UnF) and in fertilized embryos (F) performed at 1 h post-fertilization. The results are expressed as the percentage incorporation of [35S]-methionine into protein over total radioactivity taken up by the cells in three independent experiments. Error bars represent standard deviation. (C) Optical density profiles (ODA254) of polysome gradient profiles (top) and corresponding RNA profiles are shown for unfertilized eggs and fertilized embryos treated with puromycin or left untreated. The RNAs from each fraction of the polysome gradient were separated on 2% agarose-TBE gels. The positions of the 18S and 28S ribosomal RNAs are indicated. (D) Distribution on a 15â€“40% sucrose gradient of mRNAs coding for cyclin B (CycB, positive control) and initiation factor 4A (eIF4A, negative control) before (UnF) and after (F) fertilization. mRNAs were detected by RT-PCR amplification in each fraction (a representative experiment shown). Distribution of the mRNA along the gradient is shown as a percentage of total mRNA, error bars represent SEM on five biological replicates (UnF vs F: * P-value < 0.05). Presence of the mRNA in active polysomes was assessed by treating embryos in vivo with puromycin before polysome gradient fractionation (F+puro in vivo, n = 3). (E) Diagram of the translatome analysis, performed on three independent polysome profiling datasets.



	Figure 5. CDK1 protein is newly synthesized after fertilization. [35S]-methionine labeled proteins from embryos collected at the indicated times after fertilization were incubated on p13suc1-sepharose beads; affinity-purified proteins were resolved on SDS-PAGE and transferred to a nitrocellulose membrane. After exposure on a PhosphorImager screen (autoradiography), the membrane was incubated with anti-PSTAIR antibodies directed against the CDK1 protein (WB). Autoradiography and western blot on total proteins are shown in the bottom panel. Fertilized (left) and PP242-treated (right) embryos are shown. As a control, [35S]-methionine labeled embryos were cultured in presence of emetine, which was added 5 min after fertilization to inhibit protein synthesis, and the lysate prepared from embryos harvested at 90 min was used for p13suc1-sepharose bead purification (F+emet). The experiment was performed twice.
	Figure 6. Model of polysomal recruitment dynamics upon fertilization in sea urchin. Before fertilization, translation activity is generally repressed. Fertilization triggers the selective recruitment of a new subset of mRNAs onto polysomes, with over-represented functional categories such as cell cycle, RNA-binding and signaling (recruitment from free mRNAs or small mRNPs before fertilization, large black arrow). The recruitment of some mRNAs depends on the mTOR pathway, either completely or partially, whereas polysomal recruitment of other mRNAs are independent of mTOR (box). Some mRNAs present in stalled polysomes or stored in large mRNPs before fertilization are activated for translation after fertilization (thin black arrow). Fertilization also triggers the selective repression of a small subset of mRNAs translated in the egg before fertilization (dashed gray arrow), but most maternal mRNAs do not change their polysomal behavior (gray arrow).

References [+] :

Alexandraki, Expression of alpha- and beta-tubulin genes during development of sea urchin embryos. 1985, Pubmed, Echinobase