ElrA binding to the 3'UTR of cyclin E1 mRNA requires polyadenylation elements

The early cell divisions of Xenopus laevis and other metazoan embryos occur in the presence of constitutively high levels of the cell cycle regulator cyclin E1. Upon completion of the 12th cell division, a time at which many maternal proteins are downregulated by deadenylation and destabilization of their encoding mRNAs, maternal cyclin E1 protein is downregulated while its mRNA is polyadenylated and stable. We report here that stable polyadenylation of cyclin E1 mRNA requires three cis-acting elements in the 3′ untranslated region; the nuclear polyadenylation sequence, a contiguous cytoplasmic polyadenylation element and an upstream AU-rich element. ElrA, the Xenopus homolog of HuR and a member of the ELAV gene family binds the cyclin E1 3′UTR with high affinity. Deletion of these elements dramatically reduces the affinity of ElrA for the cyclin E1 3′UTR, abolishes polyadenylation and destabilizes the mRNA. Together, these findings provide compelling evidence that ElrA functions in polyadenylation and stabilization of cyclin E1 mRNA via binding these elements.     

Translational control of cyclin B1 mRNA during meiotic maturation: coordinated repression and cytoplasmic polyadenylation

Translational control is prominent during meiotic maturation and early development. In this report, we investigate a mode of translational repression in Xenopus laevis oocytes, focusing on the mRNA encoding cyclin B1. Translation of cyclin B1 mRNA is relatively inactive in the oocyte and increases dramatically during meiotic maturation. We show, by injection of synthetic mRNAs, that the cis-acting sequences responsible for repression of cyclin B1 mRNA reside within its 3'UTR. Repression can be saturated by increasing the concentration of reporter mRNA injected, suggesting that the cyclin B1 3'UTR sequences provide a binding site for a trans-acting repressor. The sequences that direct repression overlap and include cytoplasmic polyadenylation elements (CPEs), sequences known to promote cytoplasmic polyadenylation. However, the presence of a CPE per se appears insufficient to cause repression, as other mRNAs that contain CPEs are not translationally repressed. We demonstrate that relief of repression and cytoplasmic polyadenylation are intimately linked. Repressing elements do not override the stimulatory effect of a long poly(A) tail, and polyadenylation of cyclin B1 mRNA is required for its translational recruitment. Our results suggest that translational recruitment of endogenous cyclin B1 mRNA is a collaborative effect of derepression and poly(A) addition. We discuss several molecular mechanisms that might underlie this collaboration.     

Enforcing temporal control of maternal mRNA translation during oocyte cell‐cycle progression

Meiotic cell‐cycle progression in progesterone‐stimulated Xenopus oocytes requires that the translation of pre‐existing maternal mRNAs occur in a strict temporal order. Timing of translation is regulated through elements within the mRNA 3′ untranslated region (3′ UTR), which respond to cell cycle‐dependant signalling. One element that has been previously implicated in the temporal control of mRNA translation is the cytoplasmic polyadenylation element (CPE). In this study, we show that the CPE does not direct early mRNA translation. Rather, early translation is directed through specific early factors, including the Musashi‐binding element (MBE) and the MBE‐binding protein, Musashi. Our findings indicate that although the cyclin B5 3′ UTR contains both CPEs and an MBE, the MBE is the critical regulator of early translation. The cyclin B2 3′ UTR contains CPEs, but lacks an MBE and is translationally activated late in maturation. Finally, utilizing antisense oligonucleotides to attenuate endogenous Musashi synthesis, we show that Musashi is critical for the initiation of early class mRNA translation and for the subsequent activation of CPE‐dependant mRNA translation.     

Translational control of the embryonic cell cycle

The synthesis and destruction of cyclin B drives mitosis in eukaryotic cells. Cell cycle progression is also regulated at the level of cyclin B translation. In cycling extracts from Xenopus embryos, progression into M phase requires the polyadenylation-induced translation of cyclin B1 mRNA. Polyadenylation is mediated by the phosphorylation of CPEB by Aurora, a kinase whose activity oscillates with the cell cycle. Exit from M phase seems to require deadenylation and subsequent translational silencing of cyclin B1 mRNA by Maskin, a CPEB and eIF4E binding factor, whose expression is cell cycle regulated. These observations suggest that regulated cyclin B1 mRNA translation is essential for the embryonic cell cycle. Mammalian cells also display a cell cycle-dependent cytoplasmic polyadenylation, suggesting that translational control by polyadenylation might be a general feature of mitosis in animal cells.     

CPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte Maturation

Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3’UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3’UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways.     

CDK1 and calcineurin regulate Maskin association with eIF4E and translational control of cell cycle progression

Maskin regulates assembly of the eIF4F translation initiation complex on messenger RNAs that contain cytoplasmic polyadenylation elements (CPEs) in their 3' untranslated regions. Because Maskin and eIF4G contain similar peptide motifs that bind eIF4E, they compete for occupancy of this factor and consequently control translation. One mRNA that is regulated by Maskin encodes cyclin B1, whose translation oscillates with the early cell cycles of Xenopus laevis embryos. Here we show that Maskin phosphorylation-dephosphorylation also oscillates with the cell cycle and is controlled by the kinase CDK1 and the phosphatase calcineurin. These phosphorylation events control the Maskin-eIF4E interaction and, as a result, translation of cyclin B1 mRNA. Cell cycle progression requires this Maskin-mediated translational regulation.     

CPEB controls the cytoplasmic polyadenylation of cyclin, Cdk2 and c-mos mRNAs and is necessary for oocyte maturation in Xenopus.

Cytoplasmic polyadenylation is a key mechanism controlling maternal mRNA translation in early development. In most cases, mRNAs that undergo poly(A) elongation are translationally activated; those that undergo poly(A) shortening are deactivated. Poly(A) elongation is regulated by two cis-acting sequences in the 3'-untranslated region (UTR) of responding mRNAs, the polyadenylation hexanucleotide AAUAAA and the U-rich cytoplasmic polyadenylation element (CPE). Previously, we cloned and characterized the Xenopus oocyte CPE binding protein (CPEB), showing that it was essential for the cytoplasmic polyadenylation of B4 RNA. Here, we show that CPEB also binds the CPEs of G10, c-mos, cdk2, cyclins A1, B1 and B2 mRNAs. We find that CPEB is necessary for polyadenylation of these RNAs in egg extracts, suggesting that this protein is required for polyadenylation of most RNAs during oocyte maturation. Our data demonstrate that the complex timing and extent of polyadenylation are partially controlled by CPEB binding to multiple target sites in the 3' UTRs of responsive mRNAs. Finally, injection of CPEB antibody into oocytes not only inhibits polyadenylation in vivo, but also blocks progesterone-induced maturation. This is due to inhibition of polyadenylation and translation of c-mos mRNA, suggesting that CPEB is critical for early development.     

Cytoplasmic polyadenylation elements mediate masking and unmasking of cyclin B1 mRNA

During oocyte maturation, cyclin B1 mRNA is translationally activated by cytoplasmic polyadenylation. This process is dependent on cytoplasmic polyadenylation elements (CPEs) in the 3' untranslated region (UTR) of the mRNA. To determine whether a titratable factor might be involved in the initial translational repression (masking) of this mRNA, high levels of cyclin B1 3' UTR were injected into oocytes. While this treatment had no effect on the poly(A) tail length of endogenous cyclin B1 mRNA, it induced cyclin B1 synthesis. A mutational analysis revealed that the most efficient unmasking element in the cyclin 3' UTR was the CPE. However, other U-rich sequences that resemble the CPE in structure, but which do not bind the CPE-binding polyadenylation factor CPEB, failed to induce unmasking. When fused to the chloramphenical acetyl transferase (CAT) coding region, the cyclin B1 3' UTR inhibited CAT translation in injected oocytes. In addition, a synthetic 3' UTR containing multiple copies of the CPE also inhibited translation, and did so in a dose-dependent manner. Furthermore, efficient CPE-mediated masking required cap-dependent translation. During the normal course of progesterone-induced maturation, cytoplasmic polyadenylation was necessary for mRNA unmasking. A model to explain how cyclin B1 mRNA masking and unmasking could be regulated by the CPE is presented.     

Translational control of maskin mRNA by its 3' untranslated region

BACKGROUND INFORMATION: Maskin is a member of the TACC (transforming acidic coiled-coil) domain proteins found in Xenopus laevis oocytes and embryos. It has been implicated in the co-ordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. RESULTS: In the present study, we report that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3'-UTR (untranslated region) of maskin can confer the translational regulation to a reporter mRNA, and so can the 3'-UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV cross-linking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3'-UTRs. As reported previously, CPEB [CPE (cytoplasmic polyadenylation element)-binding protein] binds to the cyclin B1 3'-UTR, but its binding to the maskin 3'-UTR is minimal. By RNA affinity chromatography and MS, we identified the EDEN-BP [EDEN (embryonic deadenylation element)-binding protein] as one of the proteins binding to both the maskin and the cyclin B1 3'-UTRs. CONCLUSIONS: Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3'-UTRs, indicating it may be involved in the deadenylation of these mRNAs.     

The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes.

Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3' untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5' UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation.     

Direct binding of specific AUF1 isoforms to tandem zinc finger domains of tristetraprolin (TTP) family proteins

Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA ～5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.     

Different cyclin types collaborate to reverse the S-phase checkpoint and permit prompt mitosis

Precise timing coordinates cell proliferation with embryonic morphogenesis. As Drosophila melanogaster embryos approach cell cycle 14 and the midblastula transition, rapid embryonic cell cycles slow because S phase lengthens, which delays mitosis via the S-phase checkpoint. We probed the contributions of each of the three mitotic cyclins to this timing of interphase duration. Each pairwise RNA interference knockdown of two cyclins lengthened interphase 13 by introducing a G2 phase of a distinct duration. In contrast, pairwise cyclin knockdowns failed to introduce a G2 in embryos that lacked an S-phase checkpoint. Thus, the single remaining cyclin is sufficient to induce early mitotic entry, but reversal of the S-phase checkpoint is compromised by pairwise cyclin knockdown. Manipulating cyclin levels revealed that the diversity of cyclin types rather than cyclin level influenced checkpoint reversal. We conclude that different cyclin types have distinct abilities to reverse the checkpoint but that they collaborate to do so rapidly.     

Xenopus cold-inducible RNA-binding protein 2 interacts with ElrA,the Xenopus homolog of HuR,and inhibits deadenylation of specific mRNAs

Xenopus cold-inducible RNA-binding protein 2 (xCIRP2) is a major cytoplasmic RNA-binding protein in oocytes. In this study, we identify another RNA-binding protein ElrA, the Xenopus homolog of HuR, as an interacting protein of xCIRP2 by yeast two-hybrid screening. As ElrA stabilizes the RNA body in the in vitro mRNA stability system, we examine the role of xCIRP2 in the stabilization of mRNA and find that xCIRP2 inhibits deadenylation of AU-rich element-containing mRNA. These results suggest that xCIRP2 and ElrA may be involved in the regulation of mRNA stability at different steps. By immunoprecipitation with anti-xCIRP2 antibody, we find that xCIRP2 interacts with several mRNAs including mRNA encoding the centrosomal kinase Nek2B in oocytes. xCIRP2 also inhibits deadenylation of the mRNA substrate containing the 3'-untranslated region of Nek2B mRNA in the in vitro system. Our results suggest that xCIRP2 associates with specific mRNAs and can regulate the length of poly(A) tail in Xenopus oocytes.     

Mechanisms of translational control by the 3' UTR in development and differentiation

Translational control plays a major role in early development, differentiation and the cell cycle. In this review, we focus on the four main mechanisms of translational control by 3' untranslated regions: 1. Cytoplasmic polyadenylation and deadenylation; 2. Recruitment of 4E binding proteins; 3. Regulation of ribosomal subunit binding; 4. Post-initiation repression by microRNAs. Proteins with conserved functions in translational control during development include cytoplasmic polyadenylation element binding proteins (CPEB/Orb), Pumilio, Bruno, Fragile X mental retardation protein and RNA helicases. The translational regulation of the mRNAs encoding cyclin B1, Oskar, Nanos, Male specific lethal 2 (Msl-2), lipoxygenase and Lin-14 is discussed.