Regulated Pumilio-2 binding controls RINGO/Spy mRNA translation and CPEB activation

CPEB is a sequence-specific RNA-binding protein that controls the polyadenylation-induced translation of mos and cyclin B1 mRNAs in maturing Xenopus oocytes. CPEB activity requires not only the phosphorylation of S174, but also the synthesis of a heretofore-unknown upstream effector molecule. We show that the synthesis of RINGO/Spy, an atypical activator of cyclin-dependent kinases (cdks), is necessary for CPEB-directed polyadenylation. Deletion analysis and mRNA reporter assays show that a cis element in the RINGO/Spy 3'UTR is necessary for translational repression in immature (G2-arrested) oocytes. The repression is mediated by 3'UTR Pumilio-Binding Elements (PBEs), and by its binding protein Pumilio 2 (Pum2). Pum2 also interacts with the Xenopus homolog of human Deleted for Azoospermia-like (DAZL) and the embryonic poly(A)-binding protein (ePAB). Following the induction of maturation, Pum2 dissociates not only from RINGO/Spy mRNA, but from XDAZL and ePAB as well; as a consequence, RINGO/Spy mRNA is translated. These results demonstrate that a reversible Pum2 interaction controls RINGO/Spy mRNA translation and, as a result, CPEB-mediated cytoplasmic polyadenylation.     

CPEB regulation of human cellular senescence, energy metabolism, and p53 mRNA translation

Cytoplasmic polyadenylation element-binding protein (CPEB) stimulates polyadenylation and translation in germ cells and neurons. Here, we show that CPEB-regulated translation is essential for the senescence of human diploid fibroblasts. Knockdown of CPEB causes skin and lung cells to bypass the M1 crisis stage of senescence; reintroduction of CPEB into the knockdown cells restores a senescence-like phenotype. Knockdown cells that have bypassed senescence undergo little telomere erosion. Surprisingly, knockdown of exogenous CPEB that induced a senescence-like phenotype results in the resumption of cell growth. CPEB knockdown cells have fewer mitochondria than wild-type cells and resemble transformed cells by having reduced respiration and reactive oxygen species (ROS), normal ATP levels, and enhanced rates of glycolysis. p53 mRNA contains cytoplasmic polyadenylation elements in its 3′ untranslated region (UTR), which promote polyadenylation. In CPEB knockdown cells, p53 mRNA has an abnormally short poly(A) tail and a reduced translational efficiency, resulting in an ~50% decrease in p53 protein levels. An shRNA-directed reduction in p53 protein by about 50% also results in extended cellular life span, reduced respiration and ROS, and increased glycolysis. Together, these results suggest that CPEB controls senescence and bioenergetics in human cells at least in part by modulating p53 mRNA polyadenylation-induced translation.     

Progesterone and insulin stimulation of CPEB-dependent polyadenylation is regulated by Aurora A and glycogen synthase kinase-3

Progesterone stimulation of Xenopus oocyte maturation requires the cytoplasmic polyadenylation-induced translation of mos and cyclin B mRNAs. One cis element that drives polyadenylation is the CPE, which is bound by the protein CPEB. Polyadenylation is stimulated by Aurora A (Eg2)-catalyzed CPEB serine 174 phosphorylation, which occurs soon after oocytes are exposed to progesterone. Here, we show that insulin also stimulates Aurora A-catalyzed CPEB S174 phosphorylation, cytoplasmic polyadenylation, translation, and oocyte maturation. However, these insulin-induced events are uniquely controlled by PI3 kinase and PKC-zeta, which act upstream of Aurora A. The intersection of the progesterone and insulin signaling pathways occurs at glycogen synthase kinase 3 (GSK-3), which regulates the activity of Aurora A. GSK-3 and Aurora A interact in vivo, and overexpressed GSK-3 inhibits Aurora A-catalyzed CPEB phosphorylation. In vitro, GSK-3 phosphorylates Aurora A on S290/291, the result of which is an autophosphorylation of serine 349. GSK-3 phosphorylated Aurora A, or Aurora A proteins with S290/291D or S349D mutations, have reduced or no capacity to phosphorylate CPEB. Conversely, Aurora A proteins with S290/291A or S349A mutations are constitutively active. These results suggest that the progesterone and insulin stimulate maturation by inhibiting GSK-3, which allows Aurora A activation and CPEB-mediated translation.     

Facilitation of dendritic mRNA transport by CPEB

In neurons, the proteins derived from mRNAs localized in dendrites have been implicated in synaptic plasticity. The cytoplasmic polyadenylation element (CPE), a cis element in the 3'-UTRs of specific dendritic mRNAs, promotes cytoplasmic polyadenylation-induced translation in response to synaptic stimulation. Here, we demonstrate that the CPE and its binding protein CPEB facilitate mRNA transport to dendrites. In rat hippocampal neurons infected with recombinant viruses, the CPE is sufficient to direct a reporter RNA into dendrites. CPEB-GFP protein forms RNA-containing particles that are transported into dendrites in a microtubule-dependent fashion at an average velocity of 4-8 microm/min. Such particles also contain maskin, a CPEB-associated factor that mediates cap-dependent translational repression of CPE-containing mRNA, and the molecular motors dynein and kinesin. Overexpression of CPEB in neurons promotes the transport of CPE-containing endogenous MAP2 mRNA to dendrites, whereas overexpression of a mutant CPEB that is defective for interaction with molecular motors inhibits this transport. In neurons derived from CPEB knockout mice, the dendritic transport of a CPE-containing reporter RNA is reduced. These results suggest a mechanism whereby CPE-containing mRNAs can be transported to dendrites in a translationally dormant form, but activated at synapses in response to NMDA receptor stimulation.     

A novel embryonic poly(A) binding protein, ePAB, regulates mRNA deadenylation in Xenopus egg extracts

An in vitro system that recapitulates the in vivo effect of AU-rich elements (AREs) on mRNA deadenylation has been developed from Xenopus activated egg extracts. ARE-mediated deadenylation is uncoupled from mRNA body decay, and the rate of deadenylation increases with the number of tandem AUUUAs. A novel ARE-binding protein called ePAB (for embryonic poly(A)-binding protein) has been purified from this extract by ARE affinity selection. ePAB exhibits 72% identity to mammalian and Xenopus PABP1 and is the predominant poly(A)-binding protein expressed in the stage VI oocyte and during Xenopus early development. Immunodepletion of ePAB increases the rate of both ARE-mediated and default deadenylation in vitro. In contrast, addition of even a small excess of ePAB inhibits deadenylation, demonstrating that the ePAB concentration is critical for determining the rate of ARE-mediated deadenylation. These data argue that ePAB is the poly(A)-binding protein responsible for stabilization of poly(A) tails and is thus a potential regulator of mRNA deadenylation and translation during early development.     

CPEB4 is a candidate biomarker for defining metastatic cancers and directing personalized therapies

Identification of prognostic markers of metastatic disease and targets for treatment is critical for the management of cancer patients. Cytoplasmic polyadenylation element binding protein 4 (CPEB4) associates with specific sequences in mRNA 3′ untranslated regions and promote translation by inducing cytoplasmic polyadenylation. Aberrant expression of CPEB4 correlates with certain types of cancer, indicating that CPEB4 might play critical roles in the control of cancer proliferation and metastasis. Here we demonstrate that (A) CPEB4 is selectively overexpressed in invasive or metastatic cancers and has the potential to be used for defining cancer subtypes. (B) CPEB4 might promote invasion and metastasis by exerting its effect on TGF-beta signaling pathway. (C) By generating a CPEB4 regulated gene-drug network, we show that CPEB4 is a candidate biomarker that could be beneficial for directing therapies. Taken together, our results indicate that CPEB4 is a candidate biomarker for defining metastatic cancers and directing personalized therapies.     

Isolation of novel murine maternal mRNAs regulated by cytoplasmic polyadenylation.

The cytoplasmic polyadenylation element (CPE) is an AU-rich sequence in the 3'-untranslated region of many stored maternal mRNAs. The CPE directs the meiotic maturation-specific cytoplasmic polyadenylation and translational activation of these dormant mRNAs in Xenopus. The work presented here demonstrates that the CPE controls a similar regulation in mouse oocytes and utilizes the information to isolate novel maternal mRNAs by polymerase chain reaction (PCR). A degenerate CPE primer was used in an anchored PCR reaction with cDNAs from primary mouse oocytes. Clones were identified that contained the canonical polyadenylation signal AATAAA. A novel PCR test was then used to determine the polyadenylation state of the respective mRNAs before and after meiotic maturation. Two mRNAs, OM-1 and OM-2, are cytoplasmically polyadenylated upon maturation. Another mRNA is not polyadenylated during maturation, although it contains multiple CPE-like elements, indicating that this sequence element is not sufficient for adenylation during this time. Microinjection into primary oocytes of antisense oligodeoxynucleotides directed against OM-1 destroys the mRNA but does not appear to interfere with maturation in vitro. These experiments identify two novel maternal mRNAs and establish a simple strategy for isolating other maternal messages that control meiotic maturation, fertilization, and early mouse development.     

Control of cellular senescence by CPEB

Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16(INK4A); however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence.     

Expression of CPEB,GAPDH and U6snRNA in cervical and ovarian tissue during cancer development

Persistent infection with high‐risk human papillomavirus (HPV) and expression of the proteins E6 and E7 is a prerequisite for development of cervical cancer. The distal non‐coding part of E6/E7 messengers from several HPV types is able to downregulate synthesis of a reporter gene through mechanisms with involvement of cytoplasmic polyadenylation elements (CPEs) in the messengers. We here show that the mRNA levels of one of the four known CPE‐binding proteins (CPEBs), the CPEB3, were downregulated in HPV‐positive cervical cancers, whereas in ovarian cancer the CPEB1 mRNA level was downregulated. In addition, we showed that the RNA levels of the widely used reference marker GAPDH were upregulated in both cancer forms, and the level of the reference marker U6snRNA was upregulated in cervical cancers. Moreover, a possible correlation between the degree of U6snRNA upregulation and cervical cancer propagation was shown. These changes observed in CPEB1 and CPEB3 might indicate regulatory functions of CPEBs in cancer development of HPV‐positive and HPV‐negative tumors, respectively, and the U6snRNA, GAPDH mRNA and CPEB1 mRNA levels may be useful as tumor markers for genital cancers although further investigations are needed.     

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

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 p34cdc2 kinase in cyclin-mediated polyadenylation was demonstrated by p13-Sepharose depletion; removal of the kinase from oocyte extracts with this affinity matrix abolishes polyadenylation activation. Reintroduction of cell lysates containing baculovirus-expressed p34cdc2, 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 p34cdc2. This size shift, which is reversed by treatment with acid phosphatase, coincides temporally with cyclin-induced polyadenylation activation. We propose that p34cdc2 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.     

MicroRNP‐mediated translational activation of nonadenylated mRNAs in a mammalian cell‐free system

MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4‐NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let‐7‐microRNP‐mediated translational activation of nonadenylated target mRNAs in a mammalian cell‐free system, which contains over‐expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4‐1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)‐binding protein (PABP) interacting motif (PAM2) from the TNRC6B‐SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP‐mediated mRNA regulation.     

A novel,noncanonical mechanism of cytoplasmic polyadenylation operates in Drosophila embryogenesis

Cytoplasmic polyadenylation is a widespread mechanism to regulate mRNA translation that requires two sequences in the 3′ untranslated region (UTR) of vertebrate substrates: the polyadenylation hexanucleotide, and the cytoplasmic polyadenylation element (CPE). Using a cell-free Drosophila system, we show that these signals are not relevant for Toll polyadenylation but, instead, a “polyadenylation region” (PR) is necessary. Competition experiments indicate that PR-mediated polyadenylation is required for viability and is mechanistically distinct from the CPE/hexanucleotide-mediated process. These data indicate that Toll mRNA is polyadenylated by a noncanonical mechanism, and suggest that a novel machinery functions for cytoplasmic polyadenylation during Drosophila embryogenesis.