Meiotic maturation in Xenopus requires polyadenylation of multiple mRNAs

Abstract

Cytoplasmic polyadenylation of specific mRNAs commonly is correlated with their translational activation during development. Here, we focus on links between cytoplasmic polyadenylation, translational activation and the control of meiotic maturation in Xenopus oocytes. We manipulate endogenous c-mos mRNA, which encodes a protein kinase that regulates meiotic maturation. We determined that translational activation of endogenous c-mos mRNA requires a long poly(A) tail per se, rather than the process of polyadenylation. For this, we injected 'prosthetic' poly(A)_synthetic poly(A) tails designed to attach by base pairing to endogenous c-mos mRNA that has had its own polyadenylation signals removed. This prosthetic poly(A) tail activates c-mos translation and restores meiotic maturation in response to progesterone. Thus the role of polyadenylation in activating c-mos mRNA differs from its role in activating certain other mRNAs, for which the act of polyadenylation is required. In the absence of progesterone, prosthetic poly(A) does not stimulate c-mos expression, implying that progesterone acts at additional steps to elevate c-Mos protein. By using a general inhibitor of polyadenylation together with prosthetic poly(A), we demonstrate that these additional steps include polyadenylation of at least one other mRNA, in addition to that of c-mos mRNA. These other mRNAs, encoding regulators of meiotic maturation, act upstream of c-Mos in the meiotic maturation pathway.

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos inXenopusOocytes in Response to Progesterone

“…Barkoff et al (1998) reported that polyadenylation of mos mRNA is not sufficient for accumulation of the protein in the absence of progesterone. Moreover, when meiotic maturation is inhibited by the presence of kinase-inactive p34 cdc2 (K33R), mos mRNA is still polyadenylated in response to progesterone (Ballantyne et al, 1997) but the protein does not accumulate (Nebreda et al, 1995).…”

The Cleavage and Polyadenylation Specificity Factor in Xenopus laevis Oocytes Is a Cytoplasmic Factor Involved in Regulated Polyadenylation

“…In some instances Complete-Mini (Boehringer Mannheim) protease inhibitor cocktail was substituted for these inhibitors. Clarification of oocyte homogenates was performed as described previously (4). Proteins were separated by electrophoresis through sodium dodecyl sulfate (SDS)-6.5 to 10% polyacrylamide gel electrophoresis (PAGE) gels (25) and then transferred electrophoretically to Immobilon-P (Millipore, Bedford, Mass.)…”

A novel p34cdc2-binding and activating protein that is necessary and sufficient to trigger G2/M progression in Xenopus oocytes

“…The ability of recombinant p33 ringo to induce MPF activation and GVBD independently of protein synthesis indicates that it does not function by stimulating the translation of maternal mRNAs encoding p39 mos or other triggering proteins (Barkoff et al 1998). Moreover, the observation that p42 mpk1 activation by p33 ringo is strongly reduced by the presence of cycloheximide suggests that p33…”

“…In spite of the central role of p39 mos and the p42 mpk1 pathway in oocyte maturation, there is evidence that synthesis of other proteins in addition to p39 mos is also required for progesterone to initiate oocyte maturation (Nebreda et al 1995;Barkoff et al 1998). Thus, in many batches of oocytes, the ability of p39 mos or a constitutively active MAP kinase kinase mutant to activate pre-MPF is significantly reduced when protein synthesis is inhibited, whereas p42 mpk1 is normally activated under these conditions (Yew et al 1992;Daar et al 1993;Shibuya and Ruderman 1993;Huang et al 1995;Murakami and Vande Woude 1997).…”