Cell-free deadenylation assays with Drosophila embryo extracts

Abstract

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.