Jeske Mandy, Wahle Elmar
Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle, Germany.
Methods Enzymol. 2008;448:107-18. doi: 10.1016/S0076-6879(08)02606-2.
Deadenylation initiates degradation of most mRNAs in eukaryotes. Regulated deadenylation of an mRNA plays an important role in translation control as well, especially during animal oogenesis and early embryonic development. To investigate the mechanism of sequence-dependent deadenylation, we established an in vitro system derived from 0- to 2-h-old Drosophila embryos. These extracts faithfully reproduce several aspects of the regulation of nanos mRNA: They display translation repression and deadenylation both mediated by the same sequences within the nanos 3' UTR. Here, we describe detailed protocols for preparing Drosophila embryo extracts, and their use in deadenylation assays exemplified with exogenous RNA substrates containing the nanos 3' UTR.
去腺苷酸化启动了真核生物中大多数mRNA的降解。mRNA的调控性去腺苷酸化在翻译控制中也起着重要作用，尤其是在动物卵子发生和早期胚胎发育过程中。为了研究序列依赖性去腺苷酸化的机制，我们建立了一个源自0至2小时龄果蝇胚胎的体外系统。这些提取物忠实地再现了nanos mRNA调控的几个方面：它们表现出由nanos 3'UTR内相同序列介导的翻译抑制和去腺苷酸化。在这里，我们描述了制备果蝇胚胎提取物的详细方案，以及它们在去腺苷酸化测定中的应用，以外源RNA底物为例，该底物含有nanos 3'UTR。
