Hrg was expressed in vitro using a reticulocyte lysate expression system and its PAP activity was tested by measuring the incorporation of AMP into a primer. Substantial activity was detected in the lysate that contained Hrg, while only a very limited incorporation of [32P]-labeled AMP was recorded in the case of the control lysate. Replacement of aspartic acid by alanine at position 167 of bovine PAPII is known to disrupt the enzymatic activity of PAPII (Martin, 1996). A mutant Hrg was made with alanine instead of aspartic acid at position 175 (HrgD175A), corresponding to position 167 of bovine PAPII. This mutation results in loss of the PAP activity of Hrg, confirming that hrg encodes a counterpart of PAP in Drosophila (Murata, 2001).
The activity of Drosophila His6-tagged PAP produced in E.coli was assayed in reconstituted polyadenylation reactions. Mammalian PAP requires CPSF and PABP2 in vitro to polyadenylate specifically a pre-cleaved AAUAAA-containing RNA. The ability of Drosophila PAP to carry out polyadenylation was tested in the presence or absence of bovine CPSF and Drosophila PABP2. The RNA substrate, L3pre, was derived from the adenovirus L3 polyadenylation site. It ended at the natural cleavage site and carried a tail of ~10 A residues so that it could be bound directly by PABP2. Drosophila PAP is almost inactive on its own. PAP activity is slightly enhanced by the presence of Drosophila PABP2. Bovine CPSF stimulates PAP activity more strongly. This stimulation probably occurs as a result of CPSF tethering Drosophila PAP to the mRNA as described for mammalian PAP. In the presence of both CPSF and PABP2, Drosophila PAP generates poly(A) tails of 200- 250 nucleotides within the first minute of the reaction. This increased efficiency is probably due to enhanced processivity. After this burst, poly(A) tail extension slows as described for the bovine PAP (Wahle, 1995). These results show that Drosophila PAP produced in E.coli is active and behaves as its bovine homolog in vitro (Juge, 2002).
During the mammalian 3'-end processing reaction, PAP has been reported to be required for both the cleavage and polyadenylation steps in vitro. However, in these assays, PAP is not involved in the cleavage of all pre-mRNAs (Takagaki, 1989). Whether PAP is involved in cleavage and polyadenylation of pre-mRNAs was determined in vivo using hrg mutants. To analyse the cleavage step, RNA molecules that had not been cleaved at poly(A) sites were sought for by RT- PCR. PCR primers were selected on each side of the poly(A) sites of rp49 and sop, two ubiquitously expressed genes that encode ribosomal proteins, such that if cleavage occurs normally, no or a very low amount of PCR product is expected. Total RNA was prepared from wild-type and hrgPAP12 first instar larvae and controlled by an RT- PCR with primers located in the coding region of pgk, another gene expressed ubiquitously. In hrgPAP12, cleavage occurs normally at the poly(A) sites of rp49 and sop, as in the wild-type. As a positive control, RNA from suppressor of forked [su(f)] mutant larvae was used. su(f) encodes the Drosophila homolog of human CstF-77. The Drosophila protein is required for the cleavage step of the mRNA 3'-end processing reaction in vivo (Benoit, 2002). In the su(f) mutant, uncleaved pre-mRNAs accumulate. These RNAs can be amplified by RT-PCR for both rp49 and sop. These data indicate that, in vivo, PAP is dispensable for the cleavage step of the mRNA 3'-end processing reaction (Juge, 2002).
Poly(A) tail length of rp49 and sop was measured in hrg mutants by poly(A) test (PAT) assays, a PCR-based technique that allows amplification of poly(A) tails. In wild-type first instar larvae, the longest poly(A) tails of both rp49 and sop mRNAs were found to be up to 140 residues. In the weak hrgPAP45 mutant, poly(A) tails of rp49 mRNA are not reduced and those of sop mRNA are reduced to 50% of their length in the wild-type. In hrgPAP12 mutant larvae, poly(A) tails of both rp49 and sop are strongly reduced and reach a maximal length of 30 and 60 residues for rp49 and sop mRNAs, respectively. This suggests that poly(A) tail synthesis is affected in this mutant (Juge, 2002).
Therefore, utilization of hrg mutants led to the conclusion that in vivo PAP is dispensable for the cleavage step, but is required for poly(A) tail elongation during the mRNA 3'-end processing reaction (Juge, 2002).
The poly(A)-binding protein II (PABP2) is one of the polyadenylation factors required for proper 3'-end formation of mammalian mRNAs. Pabp2, the gene encoding the Drosophila homolog of mammalian PABP2, has been cloned by using a molecular screen to identify new Drosophila proteins with RNP-type RNA-binding domains. Sequence comparison of PABP2 from Drosophila and mammals indicates that the most conserved domains are the RNA-binding domain and a coiled-coil like domain which could be involved in protein-protein interactions. Pabp2 produces four mRNAs which result from utilization of alternative poly(A) sites and encode the same protein. Using an antibody raised against Drosophila PABP2, it has been shown that the protein accumulates in nuclei of all transcriptionally active cells throughout Drosophila development. This is consistent with a general role of PABP2 in mRNA polyadenylation. Analysis of Drosophila PABP2 function in a reconstituted mammalian polyadenylation system shows that the protein has the same functions as its bovine homolog in vitro: it stimulates poly(A) polymerase and is able to control poly(A) tail length (Benoit, 1999).
Cytoplasmic polyadenylation has an essential role in activating maternal mRNA translation during early development. In vertebrates, the reaction requires CPEB, an RNA-binding protein and the poly(A) polymerase GLD-2. GLD-2-type poly(A) polymerases form a family clearly distinguishable from canonical poly(A) polymerases (PAPs). In Drosophila, canonical PAP (Hiiragi) is involved in cytoplasmic polyadenylation with Orb, the Drosophila CPEB, during mid-oogenesis. This study shows that the female germline GLD-2 is encoded by wispy. Wispy acts as a poly(A) polymerase in a tethering assay and in vivo for cytoplasmic polyadenylation of specific mRNA targets during late oogenesis and early embryogenesis. wispy function is required at the final stage of oogenesis for metaphase of meiosis I arrest and for progression beyond this stage. By contrast, canonical PAP acts with Orb for the earliest steps of oogenesis. Both Wispy and PAP interact with Orb genetically and physically in an ovarian complex. It is concluded that two distinct poly(A) polymerases have a role in cytoplasmic polyadenylation in the female germline, each of them being specifically required for different steps of oogenesis (Benoit, 2008).
In many species, the oocyte and early embryo develop in the absence of transcription. Therefore, the first steps of development depend on maternal mRNAs and on their regulation at the level of translation, stability and localization. Regulation of mRNA poly(A) tail length is a common mechanism of translational control. Deadenylation or poly(A) tail shortening results in mRNA decay or translational repression. Conversely, poly(A) tail elongation by cytoplasmic polyadenylation results in translational activation. How the poly(A) tail length of a particular mRNA and, consequently, its level of translation are determined has been a matter of investigation for many years. It is becoming clear that poly(A) tail length results from a balance between concomitant deadenylation and polyadenylation (Benoit, 2008).
The molecular mechanisms of cytoplasmic polyadenylation have been investigated in Xenopus oocytes. The specific RNA-binding protein in the reaction is CPEB (Cytoplasmic polyadenylation element binding protein), which binds the CPE in the 3'-UTR of regulated mRNAs. Two other factors, CPSF (Cleavage and polyadenylation specificity factor) and Symplekin, are required in addition to a poly(A) polymerase. Before meiotic maturation, the polyadenylation complex also contains PARN, a deadenylase whose activity counteracts poly(A) tail elongation. At meiotic maturation, CPEB phosphorylation results in the release of PARN from the complex, thus leading to polyadenylation and translational activation (Benoit, 2008).
CPSF and Symplekin are also required for nuclear polyadenylation, a cotranscriptional reaction that leads to the synthesis of a poly(A) tail at the 3' end of all mRNAs. A canonical poly(A) polymerase (PAP) is responsible for poly(A) tail synthesis during nuclear polyadenylation. Particular isoforms of PAP were first thought to be required for cytoplasmic polyadenylation. Moreover, TPAP (Papolb - Mouse Genome Informatics), a testis-specific PAP in mouse, is cytoplasmic in spermatogenic cells and has been shown, using a Tpap knockout, to be required for cytoplasmic polyadenylation of specific mRNAs and for spermiogenesis. More recently, a new family of atypical poly(A) polymerases, the GLD-2 family, has been characterized, with a first member identified in C. elegans. GLD-2-type proteins exist in all eukaryotes, where they have different functions (Benoit, 2008).
In C. elegans, GLD-2 is required for entry into meiosis from the mitotic cycle in the gonad, and for meiosis I progression. C. elegans GLD-2 has a poly(A) polymerase activity in vitro and in vivo. In Xenopus oocytes, GLD-2 is found in the cytoplasmic polyadenylation complex, within which it directly interacts with CPEB and CPSF, and it has a poly(A) polymerase activity in vitro in the presence of the other factors of the complex. GLD-2 is in complexes with mRNAs, such as cycB1 and mos, that are regulated by cytoplasmic polyadenylation. It is thus very likely that GLD-2 plays a role in cytoplasmic polyadenylation during Xenopus meiotic maturation. However, although cytoplasmic polyadenylation of mos and cycB1 mRNAs is required for meiotic maturation, the functional role of Xenopus GLD-2 in meiotic maturation has not been addressed. Unexpectedly, although mouse GLD-2 (Papd4 - Mouse Genome Informatics) is found in oocytes at metaphases I and II, a recent study shows that oocyte maturation in GLD-2 knockout mice is not altered, demonstrating that if mouse GLD-2 acts as a poly(A) polymerase at this stage, another protein acts redundantly (Benoit, 2008 and references therein).
Cytoplasmic poly(A) tail elongation is also crucial in early embryos to activate the translation of mRNAs, including that of bicoid (bcd), which encodes the anterior morphogen. Polyadenylation and translation occur upon egg activation, a process that also induces the resumption of meiosis from the metaphase I arrest in mature oocytes, and which is triggered by egg laying, the passage of the egg through the oviduct. A link has been established between cytoplasmic polyadenylation and meiotic progression at egg activation because mutants defective for meiotic progression are also defective for poly(A) tail elongation (Benoit, 2008).
This study analyzed the function of Drosophila GLD-2 in the female germline. This protein is encoded by wispy (wisp), a gene previously identified genetically, and it therefore referred to as Wisp. Wisp has a poly(A) polymerase activity in vitro and in vivo, and it is required for poly(A) tail elongation of maternal mRNAs during late oogenesis and early embryogenesis. Wisp is required for meiotic progression in mature oocytes. A key target of Wisp during this process is cortex (cort) mRNA, which encodes a meiosis-specific activator of the anaphase-promoting complex (APC). This demonstrates the role of polyadenylation and translational activation in meiotic progression. In addition, the respective roles of conventional PAP and of Wisp in oogenesis were investigate, and PAP and Orb were shown to be involved earlier than Wisp and The. These results establish the requirement of two poly(A) polymerases for cytoplasmic polyadenylation at different steps of oogenesis (Benoit, 2008).
Two genes, CG5732 and CG15737, encoding GLD-2 homologs are present in the Drosophila genome. The corresponding proteins share the characteristics of GLD-2 family members in other species. They have a catalytic DNA polymerase β-like nucleotidyltransferase domain containing three conserved aspartic acid residues that is included in a larger conserved central domain, a PAP/25A-associated domain, and they lack an RNA-binding domain. The region that is N-terminal to the central domain is variable in size and non-conserved in the Drosophila GLD-2. Several CG5732 cDNAs described in FlyBase are from adult testis, indicating that CG5732 is expressed in this tissue. RT-PCR verified that CG5732 is not expressed in ovaries. This study focused on CG15737, which was expressed in ovaries (Benoit, 2008).
This study characterized Wisp, one of the two GLD-2-type poly(A) polymerases in Drosophila. Wisp has a function in the female germline. Wisp is a bona fide poly(A) polymerase: it has poly(A) polymerase activity in a tethering assay that depends on a conserved residue in the catalytic domain. Wisp is required for poly(A) tail lengthening of a pool of mRNAs in late stages of oogenesis. GLD-2 poly(A) polymerases do not have an RNA-binding domain; instead, they interact with RNA through their association with RNA-binding proteins. In Xenopus oocytes, GLD-2 interacts with CPEB in a complex that is active in cytoplasmic polyadenylation. Wisp interacts directly with Orb. Consistent with a role for Wisp and Orb together in an ovarian cytoplasmic polyadenylation complex, wisp mutants are dominant enhancers of a weak orb allele. In C. elegans, GLD-2 has been reported to interact with the KH-domain RNA-binding protein GLD-3, which has homology with Drosophila BicC. Although this study found Wisp and BicC together in an ovarian RNP complex, their association is mediated by RNA, suggesting that the proteins do not interact directly. It has recently been reported that BicC functions in deadenylation: BicC recruits the CCR4-NOT deadenylase complex to mRNAs. However, a role was found for BicC in poly(A) tail elongation during oogenesis (Benoit, 2008 and references therein).
In addition to its function in oogenesis, Wisp-dependent cytoplasmic polyadenylation is required for the translation of essential determinants of the anteroposterior patterning of the embryo. bcd mRNA poly(A) tail elongation was known to be required for the deployment of the Bcd gradient from the anterior pole of the embryo. This study now shows that Osk and Nos accumulation at the posterior pole also depends on Wisp. This highlights the general role of poly(A) tail length regulation in Drosophila early development (Benoit, 2008).
In Drosophila, meiosis starts in the germarium, where several cells per germline cyst enter meiotic prophase. Meiosis is then restricted to a single oocyte that remains in prophase I during most of oogenesis. Progression to metaphase I (oocyte maturation) occurs in stage 13, with maintenance of metaphase I arrest in mature stage 14 oocytes. Arrested oocytes are then activated by egg laying, which induces the resumption of meiosis (Benoit, 2008).
The earliest phenotypes in wisp-null mutant are defects in metaphase I arrest and in the progression beyond this stage. This suggests that Wisp-dependent cytoplasmic polyadenylation and translational activation are essential for meiosis during and after metaphase I (but not for oocyte maturation). Consistent with this, massive translation appears to be dispensable for the completion of meiosis, but translational activation of specific mRNAs, at least of cort, is required. cort was identified as a Wisp target: cort poly(A) tail elongation and Cort accumulation in mature oocytes require Wisp. Moreover, defects in Cort accumulation in wisp mutant oocytes result in impaired CycA destruction, an event thought to be critical for meiotic progression. Wisp regulates many mRNAs at oocyte maturation, several of which might be involved at various steps of meiosis. Identification of these specific targets will be necessary to fully unravel the role of Wisp during meiosis (Benoit, 2008).
Cytoplasmic polyadenylation has been linked to meiotic progression at egg activation given that some maternal mRNAs undergo poly(A) tail elongation at egg activation. Moreover, bcd polyadenylation is affected in mutants that are defective in meiosis, such as cort mutants. It has been proposed that the link between cytoplasmic polyadenylation and egg activation results from the inactivation of canonical PAP activity by phosphorylation via the MPF (Mitotic promoting factor: Cdc2/CycB). CycB degradation by APC-Cort would both induce meiotic progression and release PAP inactivation, leading to polyadenylation (Benoit, 2008).
This model can be adapted with results presented in this study and in the recent literature. Two waves of cytoplasmic polyadenylation occur successively, one during oocyte maturation and one at egg activation. They both depend on Wisp poly(A) polymerase. The first wave is Orb-dependent and the pathway that triggers its activation is unknown. This polyadenylation induces the synthesis of Cort (and probably other proteins), which in turn is required for the second wave of cytoplasmic polyadenylation at egg activation. Cort could act in this process through the destruction of cyclins or of other proteins more specifically involved in the regulation of the polyadenylation machinery (Benoit, 2008).
A striking result in this paper is the requirement of two poly(A) polymerases for cytoplasmic polyadenylation during oogenesis. Since the discovery of GLD-2 poly(A) polymerases, it has been assumed that these proteins were responsible for cytoplasmic polyadenylation. The current data reveal a higher level of complexity to this regulation. The phenotypes of wisp mutants indicate a function of Wisp late in oogenesis. Entry into meiosis and restriction of meiosis to one oocyte, as well as DNA condensation in the karyosome, are unaffected in wisp mutants. By contrast, orb-null mutants arrest oogenesis in the germarium, with defects in the synchronous mitoses of cystoblasts and in the restriction of meiosis to one oocyte (Huynh, 2000). This study found that orb phenotypes corresponding to early defects in oogenesis, including oocyte determination and dorsoventral patterning, are dominantly enhanced by hrg mutants, strongly suggesting that canonical PAP and Orb act together in cytoplasmic polyadenylation during the first steps of oogenesis. Because Orb forms complexes with both PAP and Wisp, the same pools of mRNAs can be regulated by the two different complexes, at different steps of oogenesis. The inclusion of one or other poly(A) polymerase could allow for different types of regulation. In addition, it is possible that the presence of both poly(A) polymerases together in the complex could be required for some step of oogenesis (Benoit, 2008).
In Xenopus, GLD-2 catalyzes polyadenylation during oocyte maturation (Barnard, 2004; Rouhana, 2005), but the enzymes involved after fertilization have not been identified. Moreover, polyadenylation at earlier stages of oogenesis remains unexplored (Benoit, 2008).
CPEB function has been addressed genetically in mouse and the defect in the female germline of Cpeb-knockout mice was found to be during prophase I. By contrast, GLD-2 expression in the oocytes appears to start at metaphase I. Moreover, no female germline defective phenotype was observed in GLD-2 knockout mice. This demonstrates some level of redundancy in poly(A) polymerase function in mouse female meiosis, and indicates that the involvement of different types of poly(A) polymerase for translational activation in oogenesis and meiotic progression is common to other species (Benoit, 2008).
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