Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein.
During Xenopus oocyte maturation, poly(A) elongation controls the translational recruitment of specific mRNAs that possess a CPE (cytoplasmic polyadenylation element). To investigate the activation of polyadenylation, we have employed oocyte extracts that are not normally competent for polyadenylation. Addition of cell lysates containing baculovirus-expressed cyclin to these extracts induces the polyadenylation of exogenous B4 RNA. The involvement of p34cdc2 kinase in cyclin-mediated polyadenylation was demonstrated by p13-Sepharose depletion; removal of the kinase from oocyte extracts with this affinity matrix abolishes polyadenylation activation. Reintroduction of cell lysates containing baculovirus-expressed p34cdc2, however, completely restores this activity. To identify factors of the polyadenylation apparatus that might be responsible for the activation, we employed UV cross-linking and identified a 58-kD protein that binds the B4 CPE in oocyte extracts. In polyadenylation-proficient egg extracts, this protein has a slower electrophoretic mobility, which suggests a post-translational modification. A similar size shift of the protein is evident in oocyte extracts supplemented with lysates containing baculovirus-expressed cyclin and p34cdc2. This size shift, which is reversed by treatment with acid phosphatase, coincides temporally with cyclin-induced polyadenylation activation. We propose that p34cdc2 kinase activity leads to the phosphorylation of the 58-kD CPE-binding protein and that this event is crucial for the cytoplasmic polyadenylation that occurs during oocyte maturation.
Paris
J
J
Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Swenson
K
K
Piwnica-Worms
H
H
Richter
J D
JD
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011061:Poly A / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017027:Protein Tyrosine Phosphatases; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins; D014981:Xenopus
D002352:Carrier Proteins; D016213:Cyclins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011061:Poly A; D004247:DNA; D016203:CDC2 Protein Kinase; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases