Piqué Maria, López José Manuel, Méndez Raúl
Centre de Regulació Genòmica, Program of Gene Expression, Barcelona, Spain.
Methods Mol Biol. 2006;322:183-98. doi: 10.1007/978-1-59745-000-3_13.
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 ribosomal recruitment. In Xenopus laevis oocytes treated with the meiosis-inducing hormone progesterone, their poly(A) tails are elongated, and the mRNAs are mobilized into polysomes. 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.
脊椎动物的发育由母系遗传的信使核糖核酸引导，这些信使核糖核酸是在非常长的卵子发生期合成的。这些休眠的信使核糖核酸通常含有短的多聚腺苷酸尾巴，并作为信使核糖核蛋白储存，阻止核糖体招募。在用诱导减数分裂的激素孕酮处理的非洲爪蟾卵母细胞中，它们的多聚腺苷酸尾巴被延长，信使核糖核酸被动员到多核糖体中。这种细胞质多聚腺苷酸化由位于信使核糖核酸3'非翻译区的顺式作用元件引导。然而，并非所有母源信使核糖核酸的细胞质多聚腺苷酸化都同时发生，而是特定信使核糖核酸的翻译激活在减数分裂和早期发育过程中以顺序方式受到调节。本章描述了将显微注射的报告信使核糖核酸和放射性标记的核糖核酸注入非洲爪蟾卵母细胞以研究通过细胞质多聚腺苷酸化进行的信使核糖核酸翻译控制的方法。细胞周期蛋白B1信使核糖核酸用于说明所描述的方法。
