Cytoplasmic mRNA Polyadenylation and Translation Assays

Vertebrate development is directed by maternally inherited messenger RNAs that are synthesized during the very long period of oogenesis. These dormant mRNAs usually contain short poly(A) tails and are stored as mRNA ribonucleoproteins that preclude ribosormal recruitment. In Xenopus laevis oocytes treated with the meiosis-inducing hormone progesterone, their poly(A) tails are elongated, and the mRNAs are mobilized into polyrsomes. This cytoplasmic polyadenylation is directed by cis -acting elements located in the 3′ untranslated region of the mRNAs. However, the cytoplasmic polyadenylation of all the maternal mRNAs does not take place at once, but rather the translational activation of specific mRNAs is regulated in a sequential manner during meiosis and early development. This chapter describes the use of microinjected reporter mRNAs and radiolabeled RNAs into Xenopus oocytes to study the mRNA translational control by cytoplasmic polyadenylation. Cyclin B1 mRNA is used to illustrate the methods described.