Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein.

Abstract

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 p34 cdc2 kinase in cyclin-mediated polyadenylation was demonstrated by pl3-Sepharose depletion; removal of the kinase from oocyte extracts with this affinity matrix abolishes polyadenylation activation. Reintroduction of cell lysates containing baculovirus-expressed p34 cd~2, 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 p34 ~dc2. This size shift, which is reversed by treatment with acid phosphatase, coincides temporally with cyclin-induced polyadenylation activation. We propose that p34 cd~2 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.

Initiation of Xenopus Oocyte Maturation by Activation of the Mitogen-activated Protein Kinase Cascade

“…The increased translation of c-mos mRNA was reported to require polyadenylation (47). The maturation-induced polyadenylation of maternal mRNA(s) could be regulated by MPF, as is the case with cyclin B mRNA (48). Then, it is possible that the MAPK cascade-induced increase in the protein level of Mos is mediated by MPF activation.…”

“…SESE-MAPKK was moderately active as compared to wild-type MAPKK. 2 To produce a more active mutant of MAPKK, a deletion (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51) near the N-terminal was introduced into Xenopus SESE-MAPKK according to the method of Mansour et al (43) using primers (5Ј-AGCCTCGAGGTTTGTCTCGGCTGTAGGG-3Ј and 5Ј-CGTCTCGAGGCTTTTCTCACCCAGAAGC-3Ј) and was ligated at the XhoI site, yielding dSESE-MAPKK. This Xenopus dSESE-MAPKK can function as a strongly active mutant in Xenopus oocytes (this study) and in COS cells.…”

“…The mechanism by which MPF regulates the induction and accumulation of Mos protein remains to be determined, although MPF is known to up-regulate translation of protein synthesis and polyadenylation of maternal mRNAs (48), which increases translation efficiency. In fact, polyadenylation-mediated increase in Mos translation is recently reported to be required for Xenopus oocyte maturation (47).…”

The 36-Kilodalton Embryonic-Type Cytoplasmic Polyadenylation Element-Binding Protein in Xenopus laevis Is ElrA, a Member of the ELAV Family of RNA-Binding Proteins

“…Second, the injection of a CPEB antibody into oocytes prevents polyadenylation in vivo (40). CPEB, which is also conserved among several invertebrates (3,20,43), is phosphorylated during maturation (12,30), although the extent to which this is important for activity of the protein is unclear (5). The specificity of the interaction of CPEB for RNA resides in three parts of the protein: the two RRMs and a cysteine-histidine region that coordinates a metal ion (15).…”

GRSF-1: a poly(A)+mRNA binding protein which interacts with a conserved G-rich element

“…Phosphorylation of RNA binding proteins has been shown to be critical for their activities in some regulated processes (76)(77)(78)(79)(80)(81)(82)(83). Amino acid sequence analysis of GRSF-1 indicated a number of phosphorylation sites.…”