Translation of mRNAs in Xenopus Oocytes

Abstract

The translational control mechanisms of native and microinjected messenger RNA (mRNA) in Xenopus oocytes involve the endogenous protein synthesis machinery and govern both the kinetics of protein accumulation and function, as well as the metabolic state of the encoding mRNA.

Keywords: protein synthesis; translation initiation factors; oocyte maturation; cell cycle; maternal mRNA

Figure 1.

Meiotic cell cycle regulation of maternal mRNA translation. (a) Growth and morphology changes during oogenesis and maturation. Stages II, III, IV, V and VI oocytes are arrested in meiotic prophase I (G2). Progesterone induces cell cycle progression to meiotic metaphase II (M) and the synthesis of new proteins that change the character of the cell. Nuclear breakdown is associated with white spot formation upon maturation (Mat). (b) Phosphorylation of eIF4E during meiotic maturation. Human eIF4E expressed in resting (VI) and progesterone‐matured (Mat) oocytes was resolved by isoelectric focusing into phosphorylated (4E‐P) and nonphosphorylated (4E) forms. (c) CPEB is bound to repressed maternal mRNAs. Progesterone induces a kinase cascade that phosphorylates eIF4E and CPEB, resulting in mRNA elongation by poly(A) polymerase (PAP) and recruitment to ribosomes.

Figure 2.

Xenopus oocytes display translation initiation modes ranging from synergistic stimulation by both mRNA cap and poly(A) tail, to cap‐independent and poly(A)‐independent initiation catalysed by eIF4Gc. (a) CPE‐containing mRNAs undergo cell cycle‐regulated release from Maskin repression and synergistic mRNA recruitment by the 5′ cap and poly(A) tail. (b) Conversely, housekeeping mRNAs translate efficiently in unstimulated oocytes. Their cycle of re‐initiation requires only the minimal eIF4Gc complex. The N‐terminus eIF4G is dispensable for their translation (Keiper and Rhoads, ). eIF2, eIF3, eIF4B and tRNA, which participate in both (a) and (b), have been omitted for clarity. It is likely that (a) and (b) represent two extremes in a spectrum of mRNAs that utilize these modes to greater or lesser extents depending upon their sequence and structure.

Figure 3.

Synthesis of endogenous and exogenous proteins in Xenopus oocytes. (a) Continuous labelling by bathing of 5 oocytes in 20 μL of buffer containing [35S]methionine results in linear incorporation of radioactivity into protein for at least 2 h. The rate of amino acid uptake is estimated from total radioactivity in washed oocytes at various times during incubation. The rate of incorporation into protein is representative of protein synthetic activity when the initial rate of uptake greatly exceeds the rate of synthesis. (b) In a time course, human eIF4E accumulates for at least 48 h following the injection of 18 ng of mRNA. (c) Oocytes display synthesis of eIF4E in proportion to the dose of injected mRNA up to 10 ng (20 fmol) during a 28 h incubation. For (b) and (c), stage VI oocytes were injected equatorially with capped and polyadenylated in vitro transcribed human eIF4E mRNA and incubated at room temperature in modified Barth saline. Lysate equivalent to a quarter of an oocyte was analysed by Western blotting with antiserum against human eIF4E. ‘*’ indicates an endogenous cross‐reacting protein.

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References

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Further Reading

Wickens M, Anderson P and Jackson RJ (1997) Life and death in the cytoplasm: messages from the 3′ end. Current Opinions in Genetics and Development 7: 220–232.

Keiper BD, Gan W and Rhoads RE (1999) Molecules in focus: Protein synthesis initiation factor 4G. International Journal of Biochemistry and Cell Biology 31: 37–41.

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Keiper, Brett D(Mar 2003) Translation of mRNAs in Xenopus Oocytes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002695]