Poly(A) polymerase and the regulation of cytoplasmic polyadenylation

Translational activation in oocytes and embryos is often regulated via incr eases in poly(A) length. Cleavage and polyadenylation specificity factor (C PSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly( A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylatio n in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first app ears in vertebrate oocytes during meiotic maturation. Data presented here s hows that complexes containing both CPSF and CPEB are present in extracts o f X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. T he two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the s ame RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA seq uence preferences are sufficient to account for the specificity of cytoplas mic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is r equired for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequ ences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadeny lation and stimulated translation similar to 40-fold; stimulation was exclu sively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenyl ation and translational stimulation required PAPs catalytic core, but did n ot require the putative CPSF interaction domain of PAP. These results demon strate that premature recruitment of PAP can cause precocious polyadenylati on and translational stimulation in the resting oocyte, and can be interpre ted to suggest that the role of other factors is to deliver PAP to the mRNA .