Kim Jong Heon, Richter Joel D
Research Institute, National Cancer Center, Goyang, Gyeonggi, South Korea.
Methods Enzymol. 2008;448:119-38. doi: 10.1016/S0076-6879(08)02607-4.
The regulation of poly(A) tail length is one important mechanism for controlling gene expression during early animal development. In Xenopus oocytes, the polyadenylation-deadenylation of several essential dormant mRNAs, including cyclin B1 mRNA, are controlled by the cis-acting cytoplasmic polyadenylation element (CPE) and the hexanucleotide AAUAAA through their associations with protein factors CPEB and CPSF, respectively. CPE-containing, as well as CPE-lacking, pre-mRNAs acquire long poly(A) tails in the nucleus; after their export to the cytoplasm, there is subsequent deadenylation of CPE-containing mRNAs that is controlled by the CPEB-associated factor PARN, a poly(A)-specific ribonuclease. In general, re-adenylation after meiotic maturation of CPE-containing mRNAs is mediated by Gld2, a poly(A) polymerase. Moreover, embryonic poly(A)-binding protein, ePAB, is required for the subsequent elongation and stabilization of the poly(A) tail against PARN and other deadenylating enzymes. In this chapter, we present detailed information for measuring CPEB-mediated cytoplasmic polyadenylation-deadenylation in Xenopus laevis oocytes and egg extracts.
多聚腺苷酸（poly(A)）尾长度的调控是动物早期发育过程中控制基因表达的一种重要机制。在非洲爪蟾卵母细胞中，包括细胞周期蛋白B1 mRNA在内的几种必需的休眠mRNA的聚腺苷酸化-去腺苷酸化过程，分别通过顺式作用的细胞质聚腺苷酸化元件（CPE）和六核苷酸AAUAAA与蛋白质因子CPEB和CPSF的结合来控制。含有CPE以及缺乏CPE的前体mRNA在细胞核中获得长的poly(A)尾；在它们输出到细胞质后，含有CPE的mRNA随后会发生去腺苷酸化，这由与CPEB相关的因子PARN（一种poly(A)特异性核糖核酸酶）控制。一般来说，含有CPE的mRNA减数分裂成熟后的再腺苷酸化由poly(A)聚合酶Gld2介导。此外，胚胎型poly(A)结合蛋白ePAB对于随后poly(A)尾抵抗PARN和其他去腺苷酸化酶的延长和稳定是必需的。在本章中，我们提供了在非洲爪蟾卵母细胞和卵提取物中测量CPEB介导的细胞质聚腺苷酸化-去腺苷酸化的详细信息。
