Embryonic poly(A)-binding protein stimulates translation in germ cells

The function of poly(A)-binding protein 1 (PABP1) in poly(A)-mediated translation has been extensively characterized. Recently, Xenopus laevis oocytes and early embryos were shown to contain a novel poly(A)-binding protein, ePABP, which has not been described in other organisms. ePABP was identified as a protein that binds AU-rich sequences and prevents shortening of poly(A) tails. Here, we show that ePABP is also expressed in X. laevis testis, suggesting a more general role for ePABP in gametogenesis. We find that ePABP is conserved throughout vertebrates and that mouse and X. laevis cells have similar tissue-specific ePABP expression patterns. Furthermore, we directly assess the role of ePABP in translation. We show that ePABP is associated with polysomes and can activate the translation of reporter mRNAs in vivo. Despite its relative divergence from PABP1, we find that ePABP has similar functional domains and can bind to several PABP1 partners, suggesting that they may use similar mechanisms to activate translation. In addition, we find that PABP1 and ePABP can interact, suggesting that these proteins may be bound simultaneously to the same mRNA. Finally, we show that the activity of both PABP1 and ePABP increases during oocyte maturation, when many mRNAs undergo polyadenylation.     

Xenopus embryonic poly(A) binding protein 2 (ePABP2) defines a new family of cytoplasmic poly(A) binding proteins expressed during the early stages of vertebrate development

We describe a new RNA binding protein from Xenopus we have named ePABP2 (embryonic poly(A) binding protein type II). Based on amino acid similarity, ePABP2 is closely related to the ubiquitously expressed nuclear PABP2 protein that directs the elongation of mRNA poly(A) tails during pre-mRNA processing. However, in contrast to known PABP2 proteins, Xenopus ePABP2 is a cytoplasmic protein that is predominantly expressed during the early stages of Xenopus development and in adult ovarian tissue. Biochemical experiments indicate ePABP2 binds poly(A) with specificity and that this binding requires the RRM domain. Mouse and human ePABP2 proteins were also identified and mouse ePABP2 expression is also confined to the earliest stages of mouse development and adult ovarian tissue. We propose that Xenopus ePABP2 is the founding member of a new class of poly(A) binding proteins expressed in vertebrate embryos. Possible roles for this protein in regulating mRNA function in early vertebrate development are discussed.     

Identification of a novel Xenopus laevis poly (A) binding protein

Poly (A) binding proteins are intimately implicated in controlling a number of events in mRNA metabolism from nuclear polyadenylation to cytoplasmic translation and stability. The known poly(A) binding proteins can be divided into three distinct structural groups (prototypes PABP1, PABPN1/PABP2 and Nab2p) and two functional families, showing that similar functions can be accomplished by differing structural units. This has prompted us to perform a screen for novel poly(A) binding proteins using Xenopus laevis. A novel poly(A) binding protein of 32 kDa (p32) was identified. Sequence analysis showed that p32 has about 50% identity to the known nuclear poly(A) binding proteins (PABPN1) but is more closely related to a group of mammalian proteins of unknown function. The expression of Xenopus laevis ePABP2 is restricted to early embryos. Accordingly, we propose that p32 is the founder member of a novel class of poly(A) binding proteins named ePABP2.     

The Xenopus ELAV protein ElrB represses Vg1 mRNA translation during oogenesis

Xenopus laevis Vg1 mRNA undergoes both localization and translational control during oogenesis. We previously characterized a 250-nucleotide AU-rich element, the Vg1 translation element (VTE), in the 3'-untranslated region (UTR) of this mRNA that is responsible for the translational repression. UV-cross-linking and immunoprecipitation experiments, described here, revealed that the known AU-rich element binding proteins, ElrA and ElrB, and TIA-1 and TIAR interact with the VTE. The levels of these proteins during oogenesis are most consistent with a possible role for ElrB in the translational control of Vg1 mRNA, and ElrB, in contrast to TIA-1 and TIAR, is present in large RNP complexes. Immunodepletion of TIA-1 and TIAR from Xenopus translation extract confirmed that these proteins are not involved in the translational repression. Mutagenesis of a potential ElrB binding site destroyed the ability of the VTE to bind ElrB and also abolished translational repression. Moreover, multiple copies of the consensus motif both bind ElrB and support translational control. Therefore, there is a direct correlation between ElrB binding and translational repression by the Vg1 3'-UTR. In agreement with the reporter data, injection of a monoclonal antibody against ElrB into Xenopus oocytes resulted in the production of Vg1 protein, arguing for a role for the ELAV proteins in the translational repression of Vg1 mRNA during early oogenesis.     

Overexpression of poly(A) binding protein prevents maturation-specific deadenylation and translational inactivation in Xenopus oocytes.

The translational regulation of maternal mRNAs is the primary mechanism by which stage-specific programs of protein synthesis are executed during early development. Translation of a variety of maternal mRNAs requires either the maintenance or cytoplasmic elongation of a 3' poly(A) tail. Conversely, deadenylation results in translational inactivation. Although its precise function remains to be elucidated, the highly conserved poly(A) binding protein I (PABP) mediates poly(A)-dependent events in translation initiation and mRNA stability. Xenopus oocytes contain less than one PABP per poly(A) binding site suggesting that the translation of maternal mRNAs could be either limited by or independent of PABP. In this report, we have analyzed the effects of overexpressing PABP on the regulation of mRNAs during Xenopus oocyte maturation. Increased levels of PABP prevent the maturation-specific deadenylation and translational inactivation of maternal mRNAS that lack cytoplasmic polyadenylation elements. Overexpression of PABP does not interfere with maturation-specific polyadenylation, but reduces the recruitment of some mRNAs onto polysomes. Deletion of the C-terminal basic region and a single RNP motif from PABP significantly reduces both its binding to polyadenylated RNA in vivo and its ability to prevent deadenylation. In contrast to a yeast PABP-dependent poly(A) nuclease, PABP inhibits Xenopus oocyte deadenylase in vitro. These results indicate that maturation-specific deadenylation in Xenopus oocytes is facilitated by a low level of PABP consistent with a primary function for PABP to confer poly(A) stability.     

Squid, Cup, and PABP55B function together to regulate gurken translation in Drosophila

During Drosophila melanogaster oogenesis, the proper localization of gurken (grk) mRNA and protein is required for the establishment of the dorsal-ventral axis of the egg and future embryo. Squid (Sqd) is an RNA-binding protein that is required for the correct localization and translational regulation of the grk message. We show that Cup and polyA-binding protein (PABP) interact physically with Sqd and with each other in ovaries. We show that cup mutants lay dorsalized eggs, enhance dorsalization of weak sqd alleles, and display defects in grk mRNA localization and Grk protein accumulation. In contrast, pAbp mutants lay ventralized eggs and enhance grk haploinsufficiency. PABP also interacts genetically and biochemically with Encore. These data predict a model in which Cup and Sqd mediate translational repression of unlocalized grk mRNA, and PABP and Enc facilitate translational activation of the message once it is fully localized to the dorsal-anterior region of the oocyte. These data also provide the first evidence of a link between the complex of commonly used trans-acting factors and Enc, a factor that is required for grk translation.     

Structure of Xenopus laevis ribosomal protein L32 and its expression during development.

cDNA clones for Xenopus laevis ribosomal protein L32 have been isolated and sequenced. The deduced amino acid sequence indicates that L32 is a basic protein of 110 amino acids, has a molecular weight of 12,603 and is homologous to the rat ribosomal protein L35. Using the cDNA clone as a probe to follow the expression of this gene during Xenopus development, it has been shown that the pattern of accumulation of this mRNA follows the one previously described for other ribosomal protein mRNAs during oogenesis and embryogenesis. The analysis of the utilization of L32 mRNA during embryogenesis shows that this is controlled by the translational regulation typical of other ribosomal protein mRNAs.     

Small nuclear U-ribonucleoproteins in Xenopus laevis development. Uncoupled accumulation of the protein and RNA components

The accumulation of protein and RNA components of small nuclear U-ribonucleoprotein particles is non-co-ordinate during oogenesis and early embryogenesis in Xenopus laevis. Northern blot hybridization of a cloned Xenopus U2-RNA gene to oocyte and embryo RNAs demonstrates that the amount of small nuclear U2-RNA per oocyte reaches a plateau early in oogenesis (at the start of yolk deposition); further accumulation is not observed in oogenesis, nor in embryogenesis until the late blastula stage. In contrast, we show by immunoblot analysis that the proteins that bind to small nuclear U-RNAs continue to be accumulated after vitellogenesis begins, reaching maximum amounts only at the end of oocyte development. No further accumulation of these proteins is seen during embryogenesis. The consequences of this non-co-ordinate synthesis of small nuclear RNA and small nuclear RNA-binding proteins are as follows: a 10- to 20-fold excess of the protein components of the small ribonucleoprotein particles over small nuclear RNA exists in large oocytes; the bulk of the protein is cytoplasmic, while the RNA is nuclear. Thus the excess protein in the cytoplasm is uncomplexed with RNA. The imbalance between protein and RNA is not corrected until the late blastula or early gastrula stages of embryogenesis, when a tenfold increase in the amount of small nuclear U2-RNA is detected. Thus the protein, but not the RNA, components of small nuclear U-ribonucleoprotein particles are stockpiled in oocytes for later use in embryonic development. During the course of these studies, we also found that there are tissue-specific differences in the Sm-antigenic proteins of X. laevis.     

Cleavage of Poly(A)-Binding Protein by Enterovirus Proteases Concurrent with Inhibition of Translation In Vitro

Many enteroviruses, members of the family Picornaviridae, cause a rapid and drastic inhibition of host cell protein synthesis during infection, a process referred to as host cell shutoff. Poliovirus, one of the best-studied enteroviruses, causes marked inhibition of host cell translation while preferentially allowing translation of its own genomic mRNA. An abundance of experimental evidence has accumulated to indicate that cleavage of an essential translation initiation factor, eIF4G, during infection is responsible at least in part for this shutoff. However, evidence from inhibitors of viral replication suggests that an additional event is necessary for the complete translational shutoff observed during productive infection. This report examines the effect of poliovirus infection on a recently characterized 3′ end translational stimulatory protein, poly(A)-binding protein (PABP). PABP is involved in stimulating translation initiation in lower eukaryotes by its interaction with the poly(A) tail on mRNAs and has been proposed to facilitate 5′-end–3′-end interactions in the context of the closed-loop translational model. Here, we show that PABP is specifically degraded during poliovirus infection and that it is cleaved in vitro by both poliovirus 2A and 3C proteases and coxsackievirus B3 2A protease. Further, PABP cleavage by 2A protease is accompanied by concurrent loss of translational activity in an in vitro-translation assay. Similar loss of translational activity also occurs simultaneously with partial 3C protease-mediated cleavage of PABP in translation assays. Further, PABP is not degraded during infections in the presence of guanidine-HCl, which blocks the complete development of host translation shutoff. These results provide preliminary evidence that cleavage of PABP may contribute to inhibition of host translation in infected HeLa cells, and they are consistent with the hypothesis that PABP plays a role in facilitating translation initiation in higher eukaryotes.     

Levels of free PABP are limited by newly polyadenylated mRNA in early Spisula embryogenesis

The poly(A) tail of eukaryotic mRNAs regulates translation and RNA stability through an association with the poly(A)-binding protein (PABP). The role of PABP in selective polyadenylation/deadenylation and translational recruitment/repression of maternal mRNAs that occurs in early development is not fully understood. Here, we report studies including UV-crosslinking and immunoblotting assays to characterise PABP in the early developmental stages of the clam Spisula solidissima. A single, 70 kDa PABP, whose sequence is highly homologous to vertebrate, yeast and plant PABPs, is detected in oocytes. The levels of clam PABP are constant in early embryogenesis, although its ability to crosslink labelled poly(A) is ‘masked’ shortly after fertilisation and remains so until the larval stage. Full RNA-binding potential of PABP in embryo lysates was achieved by brief denaturation with guanidinium hydrochloride followed by dilution for binding and crosslinking or by controlled treatment of lysates with Ca²⁺-dependent micrococcal nuclease. Masking of PABP, which accompanies cytoplasmic polyadenylation in maturing oocytes and in in vitro activated oocyte lysates, is very likely due to an association with mRNAs that bear new PABP target binding sites and thus prevent protein binding to the labelled A-rich probe. Functional implications of these findings as well as the potential application of this unmasking method to other RNA-binding proteins is discussed.     

A conserved RNA-protein complex component involved in physiological germline apoptosis regulation in C. elegans

Two conserved features of oogenesis are the accumulation of translationally quiescent mRNA, and a high rate of stage-specific apoptosis. Little is understood about the function of this cell death. In C. elegans, apoptosis occurring through a specific ;physiological' pathway normally claims about half of all developing oocytes. The frequency of this germ cell death is dramatically increased by a lack of the RNA helicase CGH-1, orthologs of which are involved in translational control in oocytes and decapping-dependent mRNA degradation in yeast processing (P) bodies. Here, we describe a predicted RNA-binding protein, CAR-1, that associates with CGH-1 and Y-box proteins within a conserved germline RNA-protein (RNP) complex, and in cytoplasmic particles in the gonad and early embryo. The CGH-1/CAR-1 interaction is conserved in Drosophila oocytes. When car-1 expression is depleted by RNA interference (RNAi), physiological apoptosis is increased, brood size is modestly reduced, and early embryonic cytokinesis is abnormal. Surprisingly, if apoptosis is prevented car-1(RNAi) animals are characterized by a progressive oogenesis defect that leads rapidly to gonad failure. Elevated germ cell death similarly compensates for lack of the translational regulator CPB-3 (CPEB), orthologs of which function together with CGH-1 in diverse organisms. We conclude that CAR-1 is of critical importance for oogenesis, that the association between CAR-1 and CGH-1 has been conserved, and that the regulation of physiological germ cell apoptosis is specifically influenced by certain functions of the CGH-1/CAR-1 RNP complex. We propose that this cell death pathway facilitates the formation of functional oocytes, possibly by monitoring specific cytoplasmic events during oogenesis.     

The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2–PAN3 and CCR4–NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases.     

Rubella virus capsid protein interacts with poly(a)-binding protein and inhibits translation

During virus assembly, the capsid proteins of RNA viruses bind to genomic RNA to form nucleocapsids. However, it is now evident that capsid proteins have additional functions that are unrelated to nucleocapsid formation. Specifically, their interactions with cellular proteins may influence signaling pathways or other events that affect virus replication. Here we report that the rubella virus (RV) capsid protein binds to poly(A)-binding protein (PABP), a host cell protein that enhances translational efficiency by circularizing mRNAs. Infection of cells with RV resulted in marked increases in the levels of PABP, much of which colocalized with capsid in the cytoplasm. Mapping studies revealed that capsid binds to the C-terminal half of PABP, which interestingly is the region that interacts with other translation regulators, including PABP-interacting protein 1 (Paip1) and Paip2. The addition of capsid to in vitro translation reaction mixtures inhibited protein synthesis in a dose-dependent manner; however, the capsid block was alleviated by excess PABP, indicating that inhibition of translation occurs through a stoichiometric mechanism. To our knowledge, this is the first report of a viral protein that inhibits protein translation by sequestration of PABP. We hypothesize that capsid-dependent inhibition of translation may facilitate the switch from viral translation to packaging RNA into nucleocapsids.     

The translational repressor Cup associates with the adaptor protein Miranda and the mRNA carrier Staufen at multiple time-points during Drosophila oogenesis

In Drosophila melanogaster, Cup acts as a translational regulator during oocyte maturation and early embryogenesis. In this report, we show that Cup associates with Miranda, an adaptor protein involved in localization of specific mRNA complexes in both neuroblasts and oocytes. miranda and cup also interact genetically, since reducing miranda activity worsens the oogenesis defects associated with different cup mutant alleles. miranda mRNA is first detected within the cytoplasm of egg chambers during early oogenesis, coincidentally with very low levels of Miranda protein. We furthermore show that Cup interacts with Staufen, a protein involved in mRNA localization during oogenesis and nervous system development, and the two proteins co-localize within the posterior cytoplasm of late oocytes. Our results substantiate the idea that Cup is a multi-functional protein cooperating with different protein partners to direct egg chamber development at multiple time-points.     

Xenopus laevis zygote arrest 2 (zar2) encodes a zinc finger RNA-binding protein that binds to the translational control sequence in the maternal Wee1 mRNA and regulates translation

Zygote arrest (Zar) proteins are crucial for early embryonic development, but their molecular mechanism of action is unknown. The Translational Control Sequence (TCS) in the 3' untranslated region (UTR) of the maternal mRNA, Wee1, mediates translational repression in immature Xenopus oocytes and translational activation in mature oocytes, but the protein that binds to the TCS and mediates translational control is not known. Here we show that Xenopus laevis Zar2 (encoded by zar2) binds to the TCS in maternal Wee1 mRNA and represses translation in immature oocytes. Using yeast 3 hybrid assays and electrophoretic mobility shift assays, Zar2 was shown to bind specifically to the TCS in the Wee1 3'UTR. RNA binding required the presence of Zn(2+) and conserved cysteines in the C-terminal domain, suggesting that Zar2 contains a zinc finger. Consistent with regulating maternal mRNAs, Zar2 was present throughout oogenesis, and endogenous Zar2 co-immunoprecipitated endogenous Wee1 mRNA from immature oocytes, demonstrating the physiological significance of the protein-RNA interaction. Interestingly, Zar2 levels decreased during oocyte maturation. Dual luciferase reporter tethered assays showed that Zar2 repressed translation in immature oocytes. Translational repression was relieved during oocyte maturation and this coincided with degradation of Zar2 during maturation. This is the first report of a molecular function of zygote arrest proteins. These data show that Zar2 contains a zinc finger and is a trans-acting factor for the TCS in maternal mRNAs in immature Xenopus oocytes.