Raul Mendez

Translational control by CPEB: a means to the end

The regulated translation of messenger RNA is essential for cell-cycle progression, establishment of the body plan during early development, and modulation of key activities in the central nervous system. Cytoplasmic polyadenylation, which is one mechanism of controlling translation, is driven by CPEB — a highly conserved, sequence-specific RNA-binding protein that binds to the cytoplasmic polyadenylation element, and modulates translational repression and mRNA localization. What are the features and functions of this multifaceted protein?

Maskin Is a CPEB-Associated Factor that Transiently Interacts with eIF-4E

In Xenopus, the CPE is a bifunctional 3 UTR sequence that maintains maternal mRNA in a dormant state in oocytes and activates polyadenylation-induced translation during oocyte maturation. Here, we report that CPEB, which binds the CPE and stimulates polyadenylation, interacts with a new factor we term maskin. Maskin contains a peptide sequence that is conserved among elF-4E-binding proteins. Affinity chromatography demonstrates that CPEB, maskin, and elF-4E reside in a complex in oocytes, and yeast two-hybrid analyses indicate that CPEB and maskin bind directly, as do maskin and elF-4E. While CPEB and maskin remain together during oocyte maturation, the maskin-elF-4E interaction is substantially reduced. The dissolution of this complex may result in the binding of elF-4E to elF-4G and the translational activation of CPE-containing mRNAs.

Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA.

Full-grown Xenopus oocytes arrest at the G2/M border of meiosis I. Progesterone breaks this arrest, leading to the resumption of the meiotic cell cycles and maturation of the oocyte into a fertilizable egg. In these oocytes, progesterone interacts with an unidentified surface-associated receptor, which induces a non-transcriptional signalling pathway that stimulates the translation of dormant c-mos messenger RNA. Mos, a mitogen-activated protein (MAP) kinase kinase kinase, indirectly activates MAP kinase, which in turn leads to oocyte maturation. The translational recruitment ofxa0c- mos and several other mRNAs is regulated by cytoplasmic polyadenylation, a process that requires two 3′ untranslated regions, the cytoplasmic polyadenylation element (CPE) and thexa0polyadenylation hexanucleotide AAUAAA. Although the signalling events that trigger c-mos mRNA polyadenylation and translation are unclear, they probably involve the activation of CPEB, the CPE binding factor. Here we show that an early site-specific phosphorylation of CPEB is essential for the polyadenylation of c-mos mRNA and its subsequent translation, and for oocyte maturation. In addition, we show that this selective, early phosphorylation of CPEB is catalysed by Eg2, a member of the Aurora family of serine/threonine protein kinases.

A Combinatorial Code for CPE-Mediated Translational Control

Cytoplasmic polyadenylation plays a key role in the translational control of mRNAs driving biological processes such as gametogenesis, cell-cycle progression, and synaptic plasticity. What determines the distinct time of polyadenylation and extent of translational control of a given mRNA, however, is poorly understood. The polyadenylation-regulated translation is controlled by the cytoplasmic polyadenylation element (CPE) and its binding protein, CPEB, which can assemble both translational repression or activation complexes. Using a combination of mutagenesis and experimental validation of genome-wide computational predictions, we show that the number and relative position of two elements, the CPE and the Pumilio-binding element, with respect to the polyadenylation signal define a combinatorial code that determines whether an mRNA will be translationally repressed by CPEB, as well as the extent and time of cytoplasmic polyadenylation-dependent translational activation.

CPEB, Maskin, and Cyclin B1 mRNA at the Mitotic Apparatus: Implications for Local Translational Control of Cell Division

In Xenopus development, the expression of several maternal mRNAs is regulated by cytoplasmic polyadenylation. CPEB and maskin, two factors that control polyadenylation-induced translation are present on the mitotic apparatus of animal pole blastomeres in embryos. Cyclin B1 protein and mRNA, whose translation is regulated by polyadenylation, are colocalized with CPEB and maskin. CPEB interacts with microtubules and is involved in the localization of cyclin B1 mRNA to the mitotic apparatus. Agents that disrupt polyadenylation-induced translation inhibit cell division and promote spindle and centrosome defects in injected embryos. Two of these agents inhibit the synthesis of cyclin B1 protein and one, which has little effect on this process, disrupts the localization of cyclin B1 mRNA and protein. These data suggest that CPEB-regulated mRNA translation is important for the integrity of the mitotic apparatus and for cell division.

Phosphorylation of CPEB by Eg2 Mediates the Recruitment of CPSF into an Active Cytoplasmic Polyadenylation Complex

The release of Xenopus oocytes from prophase I arrest is largely driven by the cytoplasmic polyadenylation-induced translation of dormant maternal mRNAs. Two cis elements, the CPE and the hexanucleotide AAUAAA, and their respective binding factors, CPEB and a cytoplasmic form of CPSF, control polyadenylation. The most proximal stimulus for polyadenylation is Eg2-catalyzed phosphorylation of CPEB serine 174. Here, we show that this phosphorylation event stimulates an interaction between CPEB and CPSF. This interaction is direct, does not require RNA tethering, and occurs through the 160 kDa subunit of CPSF. Eg2-stimulated and CPE-dependent polyadenylation is reconstituted in vitro using purified components. These results demonstrate that the molecular function of Eg2-phosphorylated CPEB is to recruit CPSF into an active cytoplasmic polyadenylation complex.

Differential mRNA translation and meiotic progression require Cdc2‐mediated CPEB destruction

Translational activation of several dormant mRNAs in vertebrate oocytes is mediated by cytoplasmic polyadenylation, a process controlled by the cytoplasmic polyadenylation element (CPE) and its binding protein CPEB. The translation of CPE‐containing mRNAs does not occur en masse at any one time, but instead is temporally regulated. We show here that in Xenopus, partial destruction of CPEB controls the temporal translation of CPE‐containing mRNAs. While some mRNAs, such as the one encoding Mos, are polyadenylated at prophase I, the polyadenylation of cyclin B1 mRNA requires the partial destruction of CPEB that occurs at metaphase I. CPEB destruction is mediated by a PEST box and Cdc2‐catalyzed phosphorylation, and is essential for meiotic progression to metaphase II. CPEB destruction is also necessary for mitosis in the early embryo. These data indicate that a change in the CPEB:CPE ratio is necessary to activate mRNAs at metaphase I and drive the cells entry into metaphase II.

Meiosis requires a translational positive loop where CPEB1 ensues its replacement by CPEB4

Meiotic progression is driven by the sequential translational activation of maternal messenger RNAs stored in the cytoplasm. This activation is mainly induced by the cytoplasmic elongation of their poly(A) tails, which is mediated by the cytoplasmic polyadenylation element (CPE) present in their 3′ untranslated regions. Although polyadenylation in prophase I and metaphase I is mediated by the CPE‐binding protein 1 (CPEB1), this protein is degraded during the first meiotic division. Thus, raising the question of how the cytoplasmic polyadenylation required for the second meiotic division is achieved. In this work, we show that CPEB1 generates a positive loop by activating the translation of CPEB4 mRNA, which, in turn, replaces CPEB1 and drives the transition from metaphase I to metaphase II. We further show that CPEB1 and CPEB4 are differentially regulated by phase‐specific kinases, generating the need of two sequential CPEB activities to sustain cytoplasmic polyadenylation during all the meiotic phases. Altogether, this work defines a new element in the translational circuit that support an autonomous transition between the two meiotic divisions in the absence of DNA replication.

A fly trap mechanism provides sequence-specific RNA recognition by CPEB proteins

Cytoplasmic changes in polyA tail length is a key mechanism of translational control and is implicated in germline development, synaptic plasticity, cellular proliferation, senescence, and cancer progression. The presence of a U-rich cytoplasmic polyadenylation element (CPE) in the 3 untranslated regions (UTRs) of the responding mRNAs gives them the selectivity to be regulated by the CPE-binding (CPEB) family of proteins, which recognizes RNA via the tandem RNA recognition motifs (RRMs). Here we report the solution structures of the tandem RRMs of two human paralogs (CPEB1 and CPEB4) in their free and RNA-bound states. The structures reveal an unprecedented arrangement of RRMs in the free state that undergo an original closure motion upon RNA binding that ensures high fidelity. Structural and functional characterization of the ZZ domain (zinc-binding domain) of CPEB1 suggests a role in both protein-protein and protein-RNA interactions. Together with functional studies, the structures reveal how RNA binding by CPEB proteins leads to an optimal positioning of the N-terminal and ZZ domains at the 3 UTR, which favors the nucleation of the functional ribonucleoprotein complexes for translation regulation.

Cytoplasmic mRNA polyadenylation and translation assays.

Vertebrate development is directed by maternally inherited messenger RNAs that are synthesized during the very long period of oogenesis. These dormant mRNAs usually contain short poly(A) tails and are stored as mRNA ribonucleoproteins that preclude ribosomal recruitment. In Xenopus laevis oocytes treated with the meiosis-inducing hormone progesterone, their poly(A) tails are elongated, and the mRNAs are mobilized into polysomes. This cytoplasmic polyadenylation is directed by cis-acting elements located in the 3 untranslated region of the mRNAs. However, the cytoplasmic polyadenylation of all the maternal mRNAs does not take place at once, but rather the translational activation of specific mRNAs is regulated in a sequential manner during meiosis and early development. This chapter describes the use of microinjected reporter mRNAs and radiolabeled RNAs into Xenopus oocytes to study the mRNA translational control by cytoplasmic polyadenylation. Cyclin B1 mRNA is used to illustrate the methods described.