Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation

Cytoplasmic polyadenylation-induced mRNA translation is a hallmark of early animal development. In Xenopus oocytes, where the molecular mechanism has been defined, the core factors that control this process include CPEB, an RNA binding protein whose association with the CPE specifies which mRNAs undergo polyadenylation; CPSF, a multifactor complex that interacts with the near-ubiquitous polyadenylation hexanucleotide AAUAAA; and maskin, a CPEB and eIF4E binding protein whose regulation of initiation is governed by poly(A) tail length. Here, we define two new factors that are essential for polyadenylation. The first is symplekin, a CPEB and CPSF binding protein that serves as a scaffold upon which regulatory factors are assembled. The second is xGLD-2, an unusual poly(A) polymerase that is anchored to CPEB and CPSF even before polyadenylation begins. The identification of these factors has broad implications for biological process that employ polyadenylation-regulated translation, such as gametogenesis, cell cycle progression, and synaptic plasticity.     

Poly(A) polymerase and the regulation of cytoplasmic polyadenylation

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.     

Human Fip1 is a subunit of CPSF that binds to U-rich RNA elements and stimulates poly(A) polymerase

In mammals, polyadenylation of mRNA precursors (pre-mRNAs) by poly(A) polymerase (PAP) depends on cleavage and polyadenylation specificity factor (CPSF). CPSF is a multisubunit complex that binds to the canonical AAUAAA hexamer and to U-rich upstream sequence elements on the pre-mRNA, thereby stimulating the otherwise weakly active and nonspecific polymerase to elongate efficiently RNAs containing a poly(A) signal. Based on sequence similarity to the Saccharomyces cerevisiae polyadenylation factor Fip1p, we have identified human Fip1 (hFip1) and found that the protein is an integral subunit of CPSF. hFip1 interacts with PAP and has an arginine-rich RNA-binding motif that preferentially binds to U-rich sequence elements on the pre-mRNA. Recombinant hFip1 is sufficient to stimulate the in vitro polyadenylation activity of PAP in a U-rich element-dependent manner. hFip1, CPSF160 and PAP form a ternary complex in vitro, suggesting that hFip1 and CPSF160 act together in poly(A) site recognition and in cooperative recruitment of PAP to the RNA. These results show that hFip1 significantly contributes to CPSF-mediated stimulation of PAP activity.     

The poly A polymerase Star-PAP controls 3'-end cleavage by promoting CPSF interaction and specificity toward the pre-mRNA

Star-PAP is a poly (A) polymerase (PAP) that is putatively required for 3'-end cleavage and polyadenylation of a select set of pre-messenger RNAs (mRNAs), including heme oxygenase (HO-1) mRNA. To investigate the underlying mechanism, the cleavage and polyadenylation of pre-mRNA was reconstituted with nuclear lysates. siRNA knockdown of Star-PAP abolished cleavage of HO-1, and this phenotype could be rescued by recombinant Star-PAP but not PAPα. Star-PAP directly associated with cleavage and polyadenylation specificity factor (CPSF) 160 and 73 subunits and also the targeted pre-mRNA. In vitro and in vivo Star-PAP was required for the stable association of CPSF complex to pre-mRNA and then CPSF 73 specifically cleaved the mRNA at the 3'-cleavage site. This mechanism is distinct from canonical PAPα, which is recruited to the cleavage complex by interacting with CPSF 160. The data support a model where Star-PAP binds to the RNA, recruits the CPSF complex to the 3'-end of pre-mRNA and then defines cleavage by CPSF 73 and subsequent polyadenylation of its target mRNAs.     

Isolation of genomic and cDNA clones encoding bovine poly(A) binding protein II.

cDNA clones for bovine poly(A) binding protein II (PAB II) were isolated. Their sequence predicts a protein of 32.8 kDa, revising earlier estimates of molecular mass. The protein contains one putative RNA-binding domain of the RNP type, an acidic N-terminal and a basic C-terminal domain. Analyses of authentic PAB II were in good agreement with all predictions from the cDNA sequence except that a number of arginine residues appeared to be post-translationally modified. Poly(A) binding protein II expressed in Escherichia coli was active in poly(A) binding and reconstitution of processive polyadenylation, including poly(A) tail length control. The cDNA clones showed a number of potential PAB II binding sites in the 3' untranslated sequence. Bovine poly(A)+RNA contained two mRNAs hybridizing to a PAB II-specific probe. Analysis of a genomic clone revealed six introns in the coding sequence. The revised molecular mass led to a demonstration of PAB II oligomer formation and a reinterpretation of earlier data concerning the protein's binding to poly(A).     

Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation

Cytoplasmic polyadenylation is one mechanism that regulates translation in early animal development. In Xenopus oocytes, polyadenylation of dormant mRNAs, including cyclin B1, is controlled by the cis-acting cytoplasmic polyadenylation element (CPE) and hexanucleotide AAUAAA through associations with CPEB and CPSF, respectively. Previously, we demonstrated that the scaffold protein symplekin contacts CPEB and CPSF and helps them interact with Gld2, a poly(A) polymerase. Here, we report the mechanism by which poly(A) tail length is regulated. Cyclin B1 pre-mRNA acquires a long poly(A) tail in the nucleus that is subsequently shortened in the cytoplasm. The shortening is controlled by CPEB and PARN, a poly(A)-specific ribonuclease. Gld2 and PARN both reside in the CPEB-containing complex. However, because PARN is more active than Gld2, the poly(A) tail is short. When oocytes mature, CPEB phosphorylation causes PARN to be expelled from the ribonucleoprotein complex, which allows Gld2 to elongate poly(A) by default.     

Characterization of the multisubunit cleavage-polyadenylation specificity factor from calf thymus.

Cleavage-polyadenylation specificity factor (CPSF) is one of five separable factors known to be required for 3' cleavage and polyadenylation of mRNA precursors in vitro. Previous studies have shown that the cleavage and poly(A) addition reactions can be uncoupled in vitro and have suggested that CPSF may be the only factor essential for both of these subreactions. Here we report the purification of CPSF to near homogeneity from calf thymus and show that the purified factor contains three polypeptides of 165, 105, and 70 kDa. These polypeptides cosediment precisely with CPSF activity, which has a sedimentation coefficient of 11.5 S. Consistent with previous reports from our laboratory, purified CPSF does not contain a detectable RNA component, indicating that it is a multisubunit protein and not a small nuclear ribonucleoprotein. Extensively purified bovine CPSF can function with human poly(A) polymerase to bring about AAUAAA-dependent poly(A) addition or with human cleavage factors to catalyze accurate 3' cleavage of a pre-mRNA substrate. UV cross-linking and gel retention analyses demonstrate that highly purified CPSF interacts with one of these cleavage factors, the multisubunit cleavage-stimulation factor, to facilitate stable binding of both to an AAUAAA-containing pre-mRNA. Likewise, evidence is presented indicating that poly(A) polymerase and CPSF can interact directly.     

Xenopus Rbm9 is a novel interactor of XGld2 in the cytoplasmic polyadenylation complex

During early development, control of the poly(A) tail length by cytoplasmic polyadenylation is critical for the regulation of specific mRNA expression. Gld2, an atypical poly(A) polymerase, is involved in cytoplasmic polyadenylation in Xenopus oocytes. In this study, a new XGld2-interacting protein was identified: Xenopus RNA-binding motif protein 9 (XRbm9). This RNA-binding protein is exclusively expressed in the cytoplasm of Xenopus oocytes and interacts directly with XGld2. It is shown that XRbm9 belongs to the cytoplasmic polyadenylation complex, together with cytoplasmic polyadenylation element-binding protein (CPEB), cleavage and polyadenylation specificity factor (CPSF) and XGld2. In addition, tethered XRbm9 stimulates the translation of a reporter mRNA. The function of XGld2 in stage VI oocytes was also analysed. The injection of XGld2 antibody into oocytes inhibited polyadenylation, showing that endogenous XGld2 is required for cytoplasmic polyadenylation. Unexpectedly, XGld2 and CPEB antibody injections also led to an acceleration of meiotic maturation, suggesting that XGld2 is part of a masking complex with CPEB and is associated with repressed mRNAs in oocytes.     

A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation

Polyadenylation of mRNA precursors by poly(A) polymerase depends on a specificity factor, CPF, recognizing the polyadenylation signal AAUAAA. This paper describes an apparently novel poly(A)-binding protein that acts as a second specificity factor, mediating the recognition of the growing poly(A) tail. A transition from a slow initiation phase of polyadenylation to rapid elongation occurs when the growing tail is long enough to serve as a binding site for the poly(A)-binding protein. Elongation of an RNA carrying a tail of 10 or more adenylate residues can occur independently of CPF. A sharp decrease in the poly(A) chain growth rate after the addition of approximately 200 adenylate residues invites speculations about a role of the poly(A)-binding protein in poly(A) tail length control.     

A multisubunit 3'' end processing factor from yeast containing poly(A) polymerase and homologues of the subunits of mammalian cleavage and polyadenylation specificity factor.

Polyadenylation is the second step in 3' end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans-acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I-Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Ptalp, which previously has been implicated in pre-tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1-1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.     

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF

Processing of the 3′ end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3′ termini is not sensitive to small changes in cpsf and su(f) dosage. Received: 6 June 1997 / Accepted: 5 November 1997     

Characterization of a Drosophila homologue of the 160-kDa subunit of the cleavage and polyadenylation specificity factor CPSF

Processing of the 3′ end of mRNA precursors depends on several proteins. The multisubunit cleavage and polyadenylation specificity factor (CPSF) is required for cleavage of the mRNA precursor as well as polyadenylation. CPSF interacts with the cleavage stimulatory factor complex (CstF), and this interaction increases the specificity of binding. Following cleavage downstream of the AAUAAA site, CPSF and poly(A) polymerase (PAP) are required for efficient polyadenylation. Recently, it has been shown that 160-kDa subunit of CPSF interacts directly with the 77-kDa subunit of CstF, which is homologous to the product encoded by the Drosophila gene su(f), and with PAP. Here we report the cloning and characterization of a Drosophila homologue of CPSF-160. The 1329-amino acid dCPSF protein exhibits about 45% and 20% sequence identity, respectively, to its mammalian and yeast counterparts over its entire length. We show that the CPSF homologue is expressed throughout development and that CPSF is essential for viability. Mutations in the cpsf gene did not alter the phenotype of homozygous su(f) mutations, suggesting that, for most genes, processing of 3′ termini is not sensitive to small changes in cpsf and su(f) dosage.     

Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase.

Specific maternal mRNAs receive poly(A) during early development as a means of translational regulation. In this report, we investigated the mechanism and control of poly(A) addition during frog oocyte maturation, in which oocytes advance from first to second meiosis becoming eggs. We analyzed polyadenylation in vitro in oocyte and egg extracts. In vivo, polyadenylation during maturation requires AAUAAA and a U-rich element. The same sequences are required for polyadenylation in egg extracts in vitro. The in vitro reaction requires at least two separable components: a poly(A) polymerase and an RNA binding activity with specificity for AAUAAA and the U-rich element. The poly(A) polymerase is similar to nuclear poly(A) polymerases in mammalian cells. Through a 2000-fold partial purification, the frog egg and mammalian enzymes were found to be very similar. More importantly, a purified calf thymus poly(A) polymerase acquired the sequence specificity seen during frog oocyte maturation when mixed with the frog egg RNA binding fraction, demonstrating the interchangeability of the two enzymes. To determine how polyadenylation is activated during maturation, we compared polymerase and RNA binding activities in oocyte and egg extracts. Although oocyte extracts were much less active in maturation-specific polyadenylation, they contained nearly as much poly(A) polymerase activity. In contrast, the RNA binding activity differed dramatically in oocyte and egg extracts: oocyte extracts contained less binding activity and the activity that was present exhibited an altered mobility in gel retardation assays. Finally, we demonstrate that components present in the RNA binding fraction are rate-limiting in the oocyte extract, suggesting that fraction contains the target that is activated by progesterone treatment. This target may be the RNA binding activity itself. We propose that in spite of the many biological differences between them, nuclear polyadenylation and cytoplasmic polyadenylation during early development may be catalyzed by similar, or even identical, components.