A novel mRNA 3' untranslated region translational control sequence regulates Xenopus Wee1 mRNA translation

Cell cycle progression during oocyte maturation requires the strict temporal regulation of maternal mRNA translation. The intrinsic basis of this temporal control has not been fully elucidated but appears to involve distinct mRNA 3′ UTR regulatory elements. In this study, we identify a novel translational control sequence (TCS) that exerts repression of target mRNAs in immature oocytes of the frog, Xenopus laevis, and can direct early cytoplasmic polyadenylation and translational activation during oocyte maturation. The TCS is functionally distinct from the previously characterized Musashi/polyadenylation response element (PRE) and the cytoplasmic polyadenylation element (CPE). We report that TCS elements exert translational repression in both the Wee1 mRNA 3′ UTR and the pericentriolar material-1 (Pcm-1) mRNA 3′ UTR in immature oocytes. During oocyte maturation, TCS function directs the early translational activation of the Pcm-1 mRNA. By contrast, we demonstrate that CPE sequences flanking the TCS elements in the Wee1 3′ UTR suppress the ability of the TCS to direct early translational activation. Our results indicate that a functional hierarchy exists between these distinct 3′ UTR regulatory elements to control the timing of maternal mRNA translational activation during oocyte maturation.     

HuD Interacts with Bdnf mRNA and Is Essential for Activity-Induced BDNF Synthesis in Dendrites

Highly specific activity-dependent neuronal responses are necessary for modulating synapses to facilitate learning and memory. We present evidence linking a number of important processes involved in regulating synaptic plasticity, suggesting a mechanistic pathway whereby activity-dependent signaling, likely through protein kinase C (PKC)-mediated phosphorylation of HuD, can relieve basal repression of Bdnf mRNA translation in dendrites, allowing for increased TrkB signaling and synaptic remodeling. We demonstrate that the neuronal ELAV family of RNA binding proteins associates in vivo with several Bdnf mRNA isoforms present in the adult brain in an activity-dependent manner, and that one member, HuD, interacts directly with sequences in the long Bdnf 3'' untranslated region (3''UTR) and co-localizes with Bdnf mRNA in dendrites of hippocampal neurons. Activation of PKC leads to increased dendritic translation of mRNAs containing the long Bdnf 3''UTR, a process that is dependent on the presence of HuD and its phosphorylation at threonine residues 149 and/or 165. Thus, we found a direct effect of HuD on regulating translation of dendritic Bdnf mRNAs to mediate local and activity-dependent increases in dendritic BDNF synthesis.     

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.     

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 by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse.

The c-mos proto-oncogene product is a key element in the cascade of events leading to meiotic maturation of vertebrate oocytes. We have investigated the role of cytoplasmic polyadenylation in the translational control of mouse c-mos mRNA and its contribution to meiosis. Using an RNase protection assay we show that optimal cytoplasmic polyadenylation of c-mos mRNA requires three cis elements in the 3' UTR: the polyadenylation hexanucleotide AAUAAA and two U-rich cytoplasmic polyadenylation elements (CPEs) located 4 and 51 nucleotides upstream of the hexanucleotide. When fused to CAT coding sequences, the wild-type 3' UTR of c-mos mRNA, but not a 3' UTR containing mutations in both CPEs, confers translational recruitment during maturation. This recruitment coincides with maximum polyadenylation. To assess whether c-mos mRNA polyadenylation is necessary for maturation of mouse oocytes, we have ablated endogenous c-mos mRNA by injecting an antisense oligonucleotide, which results in a failure to progress to meiosis II after emission of the first polar body. Such antisense oligonucleotide-injected oocytes could be efficiently rescued by co-injection of a c-mos mRNA carrying a wild-type 3' UTR. However, co-injection of a c-mos mRNA lacking functional CPEs substantially lowered the rescue activity. These results demonstrate that translational control of c-mos mRNA by cytoplasmic polyadenylation is necessary for normal development.     

Expression of cyclin B1 messenger RNA isoforms and initiation of cytoplasmic polyadenylation in the bovine oocyte

Oocytes can synthesize and store maternal mRNA in an inactive translational state until the start of in vitro maturation. Cytoplasmic polyadenylation, driven by 3'-untranslated region (UTR) cis-acting cytoplasmic polyadenylation element (CPE), is associated with translational activation of cyclin B1 mRNA during maturation. The main aim of the present study was to investigate if bovine oocyte cyclin B1 mRNA undergoes cytoplasmic polyadenylation/translation during in vitro maturation, as in other species. We have found that cyclin B1 mRNA is present in two isoforms, consisting of the same open reading frame but with different 3'-UTR lengths. Only the longest isoform (cyclin B1L) has a putative CPE sequence and other regulatory sequences, and its mRNA level decreases during early embryo development. The polyadenylation state of cyclin B1L during in vitro maturation was studied. Results demonstrated that cyclin B1L bears a relatively long poly(A) tail in germinal vesicle-stage oocytes, which is further lengthened at 10 h of maturation, before metaphase I. Interestingly, cyclin B1L bears a short poly(A) tail when the ovaries and the oocytes are transported and manipulated on ice to stop the polyadenylation process. Cytoplasmic polyadenylation most probably occurs during ovary transport in warm saline, when oocytes are still in their follicular environment. Our results also show a link between cytoplasmic polyadenylation of cyclin B1 and translation/appearance of cyclin B1 protein before in vitro maturation.     

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.     

Activity-dependent polyadenylation in neurons

Activity-dependent changes in protein synthesis modify synaptic efficacy. One mechanism that regulates mRNA translation in the synapto-dendritic compartment is cytoplasmic polyadenylation, a process controlled by CPEB, the cytoplasmic polyadenylation element (CPE)-specific RNA binding protein. In neurons, very few mRNAs are known CPEB substrates, and none appear to be responsible for the effects on plasticity that are found in the CPEB knockout mouse. These results suggest that the translation of other mRNAs is regulated by CPEB. To identify them, we have developed a functional assay based on the polyadenylation of brain-derived mRNAs injected into Xenopus oocytes, a surrogate system that carries out this 3' end processing event in an efficient manner. The polyadenylated RNAs were isolated by binding to and thermal elution from poly(U) agarose and identified by microarray analysis. Selected sequences that were positive for polyadenylation were cloned and retested for polyadenylation by injection into oocytes. These sequences were then examined for activity-dependent polyadenylation in cultured hippocampal neurons. Finally, the levels of two proteins encoded by polyadenylated mRNAs were examined in glutamate-stimulated synaptoneurosomes. These studies show that many mRNAs undergo activity-dependent polyadenylation in neurons and that this process coincides with increased translation in the synapto-dendritic compartment.     

Possible involvement of insulin-like growth factor 2 mRNA-binding protein 3 in zebrafish oocyte maturation as a novel cyclin B1 mRNA-binding protein that represses the translation in immature oocytes

In immature zebrafish oocytes, dormant cyclin B1 mRNAs localize to the animal polar cytoplasm as aggregates. After hormonal stimulation, cyclin B1 mRNAs are dispersed and translationally activated, which are necessary and sufficient for the induction of zebrafish oocyte maturation. Besides cytoplasmic polyadenylation element-binding protein (CPEB) and cis-acting elements in the 3′ untranslated region (UTR), Pumilio1 and a cis-acting element in the coding region of cyclin B1 mRNA are important for the subcellular localization and timing of translational activation of the mRNA. However, mechanisms underlying the spatio-temporal control of cyclin B1 mRNA translation during oocyte maturation are not fully understood. We report that insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), which was initially described as a protein bound to Vg1 mRNA localized to the vegetal pole of Xenopus oocytes, binds to the 3′ UTR of cyclin B1 mRNA that localizes to the animal pole of zebrafish oocytes. IMP3 and cyclin B1 mRNA co-localize to the animal polar cytoplasm of immature oocytes, but in mature oocytes, IMP3 dissociates from the mRNA despite the fact that its protein content and phosphorylation state are unchanged during oocyte maturation. IMP3 interacts with Pumilio1 and CPEB in an mRNA-dependent manner in immature oocytes but not in mature oocytes. Overexpression of IMP3 and injection of anti-IMP3 antibody delayed the progression of oocyte maturation. On the basis of these results, we propose that IMP3 represses the translation of cyclin B1 mRNA in immature zebrafish oocytes and that its release from the mRNA triggers the translational activation.     

Mos 3' UTR regulatory differences underlie species-specific temporal patterns of Mos mRNA cytoplasmic polyadenylation and translational recruitment during oocyte maturation

The Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal Mos mRNA. However, the precise temporal requirements for Mos protein function differ between oocytes of model mammalian species and oocytes of the frog Xenopus laevis. Despite the advances in model organisms, it is not known if the translation of the human Mos mRNA is also regulated by cytoplasmic polyadenylation or what regulatory elements may be involved. We report that the human Mos 3' untranslated region (3' UTR) contains a functional cytoplasmic polyadenylation element (CPE) and demonstrate that the endogenous Mos mRNA undergoes maturation-dependent cytoplasmic polyadenylation in human oocytes. The human Mos 3' UTR interacts with the human CPE-binding protein and exerts translational control on a reporter mRNA in the heterologous Xenopus oocyte system. Unlike the Xenopus Mos mRNA, which is translationally activated by an early acting Musashi/polyadenylation response element (PRE)-directed control mechanism, the translational activation of the human Mos 3' UTR is dependent on a late acting CPE-dependent process. Taken together, our findings suggest a fundamental difference in the 3' UTR regulatory mechanisms controlling the temporal induction of maternal Mos mRNA polyadenylation and translational activation during Xenopus and mammalian oocyte maturation.     

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.     

Fine mapping of interactions between eEF1α protein and 3′UTR of metallothionein-1 mRNA

The localization of metallothionein-1 (MT-1) mRNA to the perinuclear cytoskeleton is determined by a signal in the 3′untranslated region (3′UTR) and trans-acting binding proteins. The present study carried out detailed mapping of this signal and further characterized the binding to elongation factor 1 alpha (eEF1α) and other interacting proteins. Electrophoresis mobility shift assays demonstrated that shortening of a stem region proximal to nucleotides 66-76 abrogated binding. Full length recombinant rat eEF1α, and independently domains I and III, formed complexes with the mRNA. Proteins binding to biotinylated MT-1 3′UTR sequences were isolated using RNA-affinity techniques, and mass spectrometry identified histidine-tRNA ligase as one of the major MT-1 3′UTR binding proteins. We conclude that a 5-bp internal stem in the MT-1 3′UTR is critical for binding of eEF1α and histidine-tRNA ligase, and that binding of eEF1α is facilitated through domains I and III.     

The temporal control of Wee1 mRNA translation during Xenopus oocyte maturation is regulated by cytoplasmic polyadenylation elements within the 3'-untranslated region

The Wee1 protein tyrosine kinase is a key regulator of cell cycle progression. Wee1 activity is necessary for the control of the first embryonic cell cycle following the fertilization of meiotically mature Xenopus oocytes. Wee1 mRNA is present in immature oocytes, but Wee1 protein does not accumulate in immature oocytes or during the early stages of progesterone-stimulated maturation. This delay in Wee1 translation is critical since premature Wee1 protein accumulation has been shown to inhibit oocyte maturation. In this study we provide evidence that Wee1 protein accumulation is regulated at the level of mRNA translation. This translational control is directed by sequences within the Wee1 mRNA 3'-untranslated region (3' UTR). Specifically, cytoplasmic polyadenylation element (CPE) sequences within the Wee1 3' UTR are necessary for full translational repression in immature oocytes. Our data further indicate that while CPE-independent mechanisms may regulate the levels of Wee1 protein accumulation during progesterone-stimulated oocyte maturation, the timing of Wee1 mRNA translational induction is directed through a CPE-dependent mechanism.     

A novel regulatory element determines the timing of Mos mRNA translation during Xenopus oocyte maturation

Progression through vertebrate oocyte maturation requires that pre-existing, maternally derived mRNAs be translated in a strict temporal order. The mechanism that controls the timing of oocyte mRNA translation is unknown. In this study we show that the early translational induction of the mRNA encoding the Mos proto-oncogene is mediated through a novel regulatory element within the 3' untranslated region of the Mos mRNA. This novel element is responsive to the MAP kinase signaling pathway and is distinct from the late acting, cdc2-responsive, cytoplasmic polyadenylation element. Our findings suggest that the timing of maternal mRNA translation is controlled through signal transduction pathways targeting distinct 3' UTR mRNA elements.