Cytoplasmic Polyadenylation Element (CPE)- and CPE-binding Protein (CPEB)-independent Mechanisms Regulate Early Class Maternal mRNA Translational Activation in Xenopus Oocytes

Abstract

Meiotic cell cycle progression during vertebrate oocyte maturation requires the correct temporal translation of maternal mRNAs encoding key regulatory proteins. The mechanism by which specific mRNAs are temporally activated is unknown, although both cytoplasmic polyadenylation elements (CPE) within the 3 -untranslated region (3 -UTR) of mRNAs and the CPEbinding protein (CPEB) have been implicated. We report that in progesterone-stimulated Xenopus oocytes, the early cytoplasmic polyadenylation and translational activation of multiple maternal mRNAs occur in a CPEand CPEB-independent manner. We demonstrate that polyadenylation response elements, originally identified in the 3 -UTR of the mRNA encoding the Mos protooncogene, direct CPE-and CPEB-independent polyadenylation of an early class of Xenopus maternal mRNAs. Our findings refute the hypothesis that CPE sequences alone account for the range of temporal inductions of maternal mRNAs observed during Xenopus oocyte maturation. Rather, our data indicate that the sequential action of distinct 3 -UTR-directed translational control mechanisms coordinates the complex temporal patterns and extent of protein synthesis during vertebrate meiotic cell cycle progression.

Ringo/Cyclin-dependent Kinase and Mitogen-activated Protein Kinase Signaling Pathways Regulate the Activity of the Cell Fate Determinant Musashi to Promote Cell Cycle Re-entry in Xenopus Oocytes

“…The increase in PCR product length is specifically due to extension of the poly(A) tail (24,25). The primers for GST reporter mRNAs, endogenous Mos, Wee1, and cyclin A1, have been described previously (24).…”

“…Polyadenylation Assays-cDNAs for polyadenylation assays were synthesized using RNA ligation-coupled PCR as described previously (24). The increase in PCR product length is specifically due to extension of the poly(A) tail (24,25).…”

Targeted translational regulation using the PUF protein family scaffold

“…The PAT assay was performed as described with the following modifications (46). The P1 anchor primer (5′-AATATTCACCTTGATCTGAAGC-3′) was modified by 5′ phosphorylation with PNK and 3′ cordycepin according the manufacturers specifications (Promega and New England Biolabs, respectively).…”

“…Constructs for the short radiolabeled RNAs in the deadenylation assays were created by PCR-amplification using forward primer AP123 and the reverse primer ljo426 for adenylated reporters or the reverse primer AP116 (46). Steps are numbered: 1, The P1 primer was ligated to the 3′ end of total RNA by RNA ligase; 2, the P1' primer was used to make cDNA; 3, a radiolabeled gene-specific primer (P2*) was used to amplify the L1 mRNA; 4, a radiolabeled nested primer (P3*) was used to enrich the L1 product from the first PCR.…”

“…The lengths of poly(A) tails on endogenous L1 mRNAs were determined using a PCR-based poly(A) tail (PAT) assay (Fig. 6B) (46).…”