InteractiveFly: GeneBrief

Gld2: Biological Overview | References

BIOLOGICAL OVERVIEW

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 (see 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 (Kim, 2006). 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 (Kadyk, 1998). C. elegans GLD-2 has a poly(A) polymerase activity in vitro (Wang, 2002) and in vivo (Suh, 2006). 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 (Barnard, 2004). 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 (Nakanishi, 2006; Nakanishi, 2007; Benoit, 2008 and references therein).

In Drosophila, poly(A) tail regulation by deadenylation and cytoplasmic polyadenylation is essential for controlling mRNAs involved in axis patterning and other aspects in early development. In ovaries, cytoplasmic polyadenylation regulates the translation of oskar (osk), the posterior determinant, and of CycB mRNAs, and this polyadenylation depends on Orb, the Drosophila homolog of CPEB. Orb is required at the earliest steps of oogenesis for the regulation of the synchronous divisions of a cystoblast that lead to the production of sixteen germ cells per cyst, and for the restriction of meiosis to one oocyte. A single gene, hiiragi (hrg), which encodes one isoform of canonical PAP, exists in the Drosophila genome. Genetic interactions have implicated orb and hrg in the cytoplasmic polyadenylation of osk mRNA and accumulation of Osk protein at the posterior pole of the oocyte during mid-oogenesis. This led to the conclusion that canonical PAP has a role in cytoplasmic polyadenylation at this stage (Benoit, 2008).

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 (Brent, 2000), 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 (Barnard, 2004; Rouhana, 2005). 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 (Chicoine, 2007). However, a role was found for BicC in poly(A) tail elongation during oogenesis (Chicoine, 2007; 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 (Pesin, 2007; Vardy, 2007). 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 (Nakanishi, 2006). Moreover, no female germline defective phenotype was observed in GLD-2 knockout mice (Nakanishi, 2007). 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).

Adenylation of maternally inherited microRNAs by Wispy

Early development depends heavily on accurate control of maternally inherited mRNAs, and yet it remains unknown how maternal microRNAs are regulated during maternal-to-zygotic transition (MZT). This study found that maternal microRNAs are highly adenylated at their 3' ends in mature oocytes and early embryos. Maternal microRNA adenylation is widely conserved in fly, sea urchin, and mouse. This study identified Wispy, a noncanonical poly(A) polymerase, as the enzyme responsible for microRNA adenylation in flies. Knockout of wispy abrogates adenylation and results in microRNA accumulation in eggs, whereas overexpression of Wispy increases adenylation and reduces microRNA levels in S2 cells. Wispy interacts with Ago1 through protein-protein interaction, which may allow the effective and selective adenylation of microRNAs. Thus, adenylation may contribute to the clearance of maternally deposited microRNAs during MZT. This work provides mechanistic insights into the regulation of maternal microRNAs and illustrates the importance of RNA tailing in development. (Lee, 2014).

This study provides mechanistic insights into the regulation of maternally deposited miRNAs. The majority of maternal miRNAs are subject to adenylation during early development in Drosophila. The sequencing and northern blotting data indicate that the levels of adenylated miRNAs begin to drop at ~1.5 hr AEL with an average half-life of ~2 hr. This is substantially shorter than those estimated in mammalian cell lines (>100 hr considering dilution caused by cell division). The results collectively suggest that Wisp induces adenylation and facilitates miRNA downregulation. Thus, miRNA adenylation may provide a molecular basis for the clearance of maternally inherited miRNAs during MZT. MiRNA tailing is often associated with miRNA destabilization. An interesting example is poxvirus whose adenylyl transferase VP55 downregulates host miRNAs. It is conceivable that poxviruses have adopted the cellular adenylation/decay machineries to their own benefits. It is noted that adenylation may have an opposing effect in different contexts, as mammalian poly(A) polymerase GLD2 is known to stabilize certain miRNAs. It is yet unclear why the seemingly related modifications result in such different consequences. To dissect the mechanism behind the link between tailing and decay, it will be important to identify the enzyme that executes decay. Exoribonuclease candidates including exosome components and a known fly miRNA trimming factor, Nibbler, were tested by individual knockdown in S2 cells, it was not possible to the enzyme(s), implying that multiple factors may act redundantly, as in Arabidopsis (Lee, 2014).

It is intriguing that three distant species (fly, sea urchin, and mouse) show a similar pattern of miRNA expression and adenylation. In all species examined, relative contents of miRNA are low in mature oocytes. In frogs and zebrafish, the miRNA proportion was particularly small in oocytes and early embryos. Thus, maternal miRNAs may be cleared out at an early stage(s) of oocyte maturation in frogs and fish so that maternal miRNAs may not be transmitted to the next generation. In flies, maternal miRNA clearance is relatively delayed and overlaps with zygotic transcription. Therefore, while the clearance of maternal miRNA appears to be a universal phenomenon, the precise timing of the event varies among species. Consistently, previous studies have suggested that miRNAs may be inactive in oocytes and early development. Maternal and zygotic Dgcr8 null mouse embryos develop normally to the blastocyst stage, indicating that miRNA function is suppressed in oocytes and early embryos. In parallel, reporter assays for endogenous miRNA activity in mouse oocytes revealed a dramatic decrease in miRNA activity along oocyte maturation. The current data suggest that active clearance of miRNA via adenylation may be necessary for normal gene regulation in early embryo development (Lee, 2014).

Wisp physically associates with Ago1, which may explain why miRNAs are effectively adenylated. However, adenylation frequency varies among miRNA species, and does not show a strong correlation with abundance. Common features were sought among the highly adenylated miRNAs, but fno sequence motif was found that is significantly enriched in adenylated miRNAs. When miR-312 (that is highly adenylated in embryos) and miR-286 (that is not adenylated in embryos) were ectopically in S2 cells, both miRNAs were similarly adenylated by Wisp, suggesting that adenylation is not determined by intrinsic sequences of miRNA. Temporal differential expression was examined in embryos. Due to an inevitable normalization issue, the heatmap does not strictly reflect the changes in absolute abundance. Nevertheless, this analysis was useful to classify miRNAs according to their expression patterns during development. MiRNAs are clustered into three groups. The 'early' miRNAs show higher expression at early stage and decline after major zygotic genome activation (~1.5 hr AEL), indicating that these miRNAs are reduced during MZT. The 'late' miRNAs are mainly induced after zygotic activation in the later stage. The 'biphasic' miRNAs also show the highest levels in late embryos, but they are detected in early stage to some degrees. By comparing adenylation ratios of three groups, it was found that the 'early' group tends to be more frequently adenylated than the other groups. This result was validated by northern blotting of two representative miRNAs. miR-312 (an 'early' miRNA) is highly adenylated and gradually disappears, while miR-286 (a 'late' miRNA) is not modified and is induced zygotically. The data collectively suggest an intriguing possibility that 'early' maternal miRNAs are downregulated during MZT, although the mechanism underlying the preference of adenylation is unclear at this point (Lee, 2014).

It is conceivable that miRNA targets may affect adenylation rate in vivo. MiRNAs bound to complementary targets may be more susceptible to Wisp-mediated adenylation because the 3' end of guide miRNA is thought to be released from the PAZ domain of AGO protein when the miRNA binds to a highly complementary target. Not mutually exclusively, some miRNAs may be more accessible to Wisp than others in vivo. Certain miRNAs may be localized separately from Wisp, and the physical segregation may protect the miRNAs from adenylation and decay. It will be interesting in the future to dissect the mechanism underlying the selectivity of adenylation (Lee, 2014).

Knockout mutation of wisp results in defects in late meiosis and early development. MiRNA adenylation activity of Wisp may at least partly contribute to the phenotype, although currently it is not possible to separate the contribution of miRNA adenylation from that of mRNA polyadenylation. Cytoplasmic polyadenylation of mRNA induces translation, while miRNA adenylation facilitates miRNA decay. Because both events are expected to positively regulate protein synthesis, it will be interesting to ask if the two seemingly independent activities corroborate to enhance translation (Lee, 2014).

Thus far, the extensive miRNA adenylation in oocytes (over 30%) is unprecedented among uninfected cell types. MiRNAs typically show less than 2%-4% of adenylation. But it is conceivable that adenylation may be used for gene regulation in diverse cellular contexts. Some specific cell types and/or subcellular locations may contain highly adenylated miRNA species. It will be interesting in the future to investigate miRNA tailing in various cell types, such as in neurons, as cytoplasmic polyadenylation is known to be active and important for local translation in neural synapses (Lee, 2014).

The poly(A) polymerase GLD2 is required for spermatogenesis in Drosophila melanogaster

The DNA of a developing sperm is normally inaccessible for transcription for part of spermatogenesis in many animals. In Drosophila melanogaster, many transcripts needed for late spermatid differentiation are synthesized in pre-meiotic spermatocytes, but are not translated until later stages. Thus, post-transcriptional control mechanisms are required to decouple transcription and translation during spermatogenesis. In the female germline, developing germ cells accomplish similar decoupling through poly(A) tail alterations to ensure that dormant transcripts are not prematurely translated: a transcript with a short poly(A) tail will remain untranslated, whereas elongating the poly(A) tail permits protein production. In Drosophila, the ovary-expressed cytoplasmic poly(A) polymerase WISPY is responsible for stage-specific poly(A) tail extension in the female germline. This study examined the possibility that a recently derived testis-expressed WISPY paralog, GLD2, plays a similar role in the Drosophila male germline. It was shown that knockdown of Gld2 transcripts causes male sterility, as GLD2-deficient males do not produce mature sperm. Spermatogenesis up to and including meiosis appears normal in the absence of GLD2, but post-meiotic spermatid development rapidly becomes abnormal. Nuclear bundling and F-actin assembly are defective in GLD2 knockdown testes and nuclei fail to undergo chromatin reorganization in elongated spermatids. GLD2 also affects the incorporation of protamines and the stability of dynamin and transition protein transcripts. The results indicate that GLD2 is an important regulator of late spermatogenesis and is the first example of a Gld-2 family member that plays a significant role specifically in male gametogenesis (Sartain, 2011).

Spermatogenesis is a tightly controlled developmental process that requires the stage-specific production of proteins. In animals, spermatogenesis begins when a diploid germline cell produced from the testis stem cell niche undergoes differentiation and proliferation though mitosis and meiosis to form many haploid spermatocytes. Post-meiotic development, called spermiogenesis, is a series of morphological changes that will determine the final shape and form of the mature sperm, which can vary greatly among taxa. One important phenomenon that is seen in spermatogenesis in many species is that transcription is silenced for part of the process: for example, transcription cannot occur after nuclear condensation in mice and there is some evidence for transcriptional silencing during meiosis in Drosophila. In such cases, any proteins that must be translated during the transcriptionally silent period must be synthesized from mRNAs that were transcribed earlier but remain untranslated until the appropriate stage of development. Furthermore, some transcripts needed for late spermiogenesis, such as those of don juan and Mst87F, are synthesized in spermatocytes, although they are not translated until much later (Sartain, 2011 and references therein).

Spermatogenesis in Drosophila melanogaster is well described. Testis gonial cells originating from germline stem cell divisions undergo synchronous mitosis and meiosis with incomplete cytokinesis, resulting in a cyst of 64 round, haploid spermatids after the completion of meiosis. The spermatids undergo morphological changes, including flagellum extension and nuclear reshaping within the syncytium, until spermatid individualization occurs. The cells exit the testis as mature sperm (Sartain, 2011).

During the final stages of spermatogenesis in Drosophila, as in many other invertebrate and vertebrate species, chromatin reorganization events cause the spermatid nuclei to become tightly compacted. Histones associated with spermatocyte chromatin are ultimately exchanged for protamines, allowing the nucleus to condense up to 200 fold. Two genes encoding protamines have been identified in Drosophila (Mst35Ba, or Protamine A; and Mst35Bb, or Protamine B). Additionally, the gene Tpl94D demonstrates functional homology to mammalian transition proteins, which bind chromatin as an intermediate step between histone-based and protamine-based chromatin organization. Therefore, nuclear compaction in Drosophila occurs as a two-step process: histones are first displaced by transition proteins, and transition proteins are later exchanged for protamines (Sartain, 2011 and references therein).

Soon after protamine incorporation, the spermatids in a cyst become separated from one another in a process called individualization. During this process, a cone-like structure composed of cross-linked F-actin assembles around each nucleus in the cyst. The 64 cones in the cyst move as a unit down the length of the sperm tails, simultaneously pushing out excess cytoplasm and wrapping each spermatid in an individual membrane. The separated, mature sperm then roll into coils and exit the testis to be stored in the seminal vesicle (Sartain, 2011).

In Drosophila, there are many examples of transcripts that are synthesized in spermatocytes but are not translated until after meiosis, to such an extent that transcriptional activity in the developing Drosophila sperm cell was previously thought to be predominantly limited to early spermatocytes and spermatogonia. However, recent evidence demonstrates that transcriptional activity occurs post-meiotically as well (Barreau, 2008; Vibranovski, 2010). For those transcripts that remain quiescent until post-meiotic stages, a translational control mechanism must be in place (Sartain, 2011).

Many cell types, including oocytes and neurons, achieve translational regulation through adjusting the length of the poly(A) tail in the cytoplasm. A long poly(A) tail promotes translation of the transcript through recruitment of translation initiation factors, whereas a transcript with a short poly(A) tail remains untranslated or is degraded. Most mRNAs are extensively polyadenylated in the nucleus; however, for some transcripts that will be held in an untranslated state for a period of time, poly(A) tail modifications occur outside the nucleus. In Xenopus, transcripts destined for post-transcriptional poly(A) tail adjustment contain two consensus sequences in their 3'UTR: a cytoplasmic polyadenylation element (CPE) and the hexamer AAUAAA; these recruit a complex of proteins that alter poly(A) tail length. In Xenopus, CPE is bound by CPE-binding protein (CPEB) (Kim, 2007). The cleavage and polyadenylation specificity factor (CPSF) binds to the hexamer. CPEB and CPSF recruit a cytoplasmic poly(A) polymerase (PAP) and a deadenylase, both of which work on the transcript simultaneously. However, the deadenylase is slightly more efficient than the PAP, so the net effect is a poly(A) tail that remains short. Upon a signal to activate translation, CPEB is phosphorylated, causing the deadenylase to dissociate from the complex; the PAP is then free to elongate the poly(A) tail (Sartain, 2011).

PAPs that act in the cytoplasmic complex differ from nuclear PAPs. The Gld-2 (germline development 2) family of cytoplasmic PAPs has been described in C. elegans, Xenopus and Drosophila. Whereas nuclear PAPs contain a catalytic domain and an RNA-binding domain, Gld-2 family members have only a catalytic domain (Bard, 2000; Martin, 2000). For RNA specificity, Gld-2 associates with an RNA-binding protein, typically a Gld-3, to form a heterodimer that acts as a cytoplasmic PAP (Wang, 2002; Sartain, 2011 and references therein).

Gld-2 family members have been shown to play roles in oogenesis in several organisms. In worms, a Gld-2 homolog is involved in the mitosis/meiosis decision to make both male and female germ cells (Kadyk, 1998). In Drosophila, the X-linked Gld-2 homolog wispy (wisp) is necessary for oogenesis and egg activation (Benoit, 2008; Cui, 2008). WISP is present in ovaries but not testes and is necessary for the completion of meiosis in oocytes. WISP has been shown to polyadenylate transcripts of cortex, which is required for proper meiotic progression (Benoit, 2008). WISP also polyadenylates several developmental transcripts, the protein products of which are needed for early embryogenesis, including bicoid, Toll and torso (Benoit, 2008; Cui, 2008; Sartain, 2011 and references therein).

The Drosophila genome contains an autosomal paralog of wisp called Gld2. Previous studies of Gld2 have demonstrated a role in long-term memory and show that GLD2 acts as a PAP in vitro (Kwak, 2008). This study shows that Gld2 is expressed in the male, but not female, germline. It is required for the completion of spermatogenesis, specifically for the elongation and individualization stages. In GLD2 knockdown testes, the first disruption observed is post-meiotic, at the onset of spermatocyst elongation. In these testes, the nuclei in developing cysts scatter and basal bodies are not observed near nuclei. F-actin-containing individualization complexes do not assemble and nuclear compaction does not complete. Additionally, protamines are not incorporated and transcripts for both dynamin (shibire - FlyBase) and the transition protein are undetectable. These findings indicate that Gld2 arose from duplication of the wisp locus, and that this derived paralog was likely maintained in the genome owing to its essential role in spermatogenesis (Sartain, 2011).

The genus Drosophila can be divided into subgenera, and genome sequences are available for two of these: Sophophora (which contains D. melanogaster) and Drosophila (which contains species that diverged from D. melanogaster ~63 million years ago). wisp and Gld2 orthologs have been identified in species from both subgenera. Available gene expression data show that the wisp orthologs in D. melanogaster, D. mojavensis and D. virilis have female-biased expression, whereas the Gld2 orthologs from D. melanogaster, D. simulans, D. sechellia, D. yakuba, D. pseudoobscura and D. mojavensis have male-biased expression (Zhang, 2007). This is consistent with the conservation of the oogenesis and spermatogenesis functions of WISP (Benoit, 2008; Cui, 2008) and GLD2, respectively, across the entire genus. All other insect species with sequenced genomes (including Anopheles gambiae and Aedes aegypti, which are the closest relatives to Drosophila with completely sequenced genomes) possess only a single ortholog of wisp/Gld2, based on best reciprocal BLAST searches. Therefore, a duplication event occurred after the most recent common ancestor (MRCA) of Drosophila and mosquitoes, but prior to the MRCA of the genus Drosophila, to give rise to either wisp or Gld2 (depending on which locus is ancestral and which is derived). A phylogenetic reconstruction of the evolutionary relationships of the insect protein coding sequences supports this hypothesis. Furthermore, the wisp/Gld2 ortholog in A. gambiae has female-biased expression, based on microarray data from whole males and females. This has led to a hypothesis that the ancestral germline function of this gene family is in ovaries, and that the testis function of GLD2 is derived (Sartain, 2011).

The testis-expressed cytoplasmic PAP, GLD2, is required for spermatogenesis in D. melanogaster. Knockdown of GLD2 in the testes causes widespread defects in post-meiotic spermatogenesis events. In a GLD2 knockdown, the earliest defects are seen in early post-meiotic spermatids, when the basal body fails to dock at the nuclear envelope and the nuclei begin to scatter. Many late-stage events of spermatogenesis are also affected, including protamine translation and F-actin cone formation on individualization stage spermatids. Additionally, GLD2 knockdown affects the stability of dynamin transcripts and those of transition protein (Tpl94D) in the testes. Interestingly, GLD2-deficient germ cells appear to undergo normal meiosis, in contrast to mutants of other Gld-2 homologs, including the GLD2 paralog WISP (Benoit, 2008; Cui, 2008; Sartain, 2011).

There is indirect evidence that GLD2 acts as a cytoplasmic PAP in the Drosophila testes. The GLD2 protein contains a PAP/25A domain, which is shared by all known Gld-2 family proteins (Benoit, 2008). Additionally, GLD2 has the ability to elongate poly(A) tails in vitro (Kwak, 2008). The current study has shown that GLD2 interacts with the Gld-3 homolog BIC-C in a yeast two-hybrid assay. Furthermore, at least two transcripts are absent in GLD2 knockdown testes, which might be the result of destabilization owing to an inability to elongate their poly(A) tails. Taken together, it is concluded that Drosophila GLD2 does act as a PAP during spermatogenesis and that the defects seen in its absence are the result of failure of one or more polyadenylation events (Sartain, 2011).

GLD2 affects many aspects of spermatogenesis in Drosophila. First, nuclear anchoring and basal body docking are defective in the absence of GLD2. Both processes occur in early post-meiotic stages of spermatogenesis in wild-type testes; however, in the absence of GLD2, spermatid nuclei scatter throughout spermatogenic cysts and basal bodies cannot assemble at the nuclear envelope. It is possible that, in the GLD2 knockdown, the failure of these events is related. For example, it might be the case that nuclear anchoring cannot occur until basal bodies have docked properly, or vice versa. Alternatively, loss of GLD2 might affect formation of the post-meiotic nuclear envelope, which might in turn have negative effects on both nuclear anchoring and basal body docking processes. Other studies of mutants that involve basal body defects have documented nuclear localization disruptions, indicating that these two processes may be linked (Sartain, 2011).

Second, GLD2 knockdown testes show abnormalities during nuclear condensation: nuclei clearly begin to condense, but condensation stalls at the canoe stage and does not progress further. It is believed that this phenotype reflects, at least in part, the absence of Tpl94D transition protein transcripts in GLD2 knockdown testes, and thus the inability of nuclei to progress to a condensation state at which protamines would be incorporated. In addition, and perhaps contributing to the phenotype, Protamine B is not translated in the absence of GLD2, even though its RNA is present. Thus, GLD2 acts upstream of Protamine B translation. Given that no effects were detected of GLD2 knockdown on the poly(A) tail length of protamine transcripts, it seems likely that removing GLD2 causes a block in the spermatogenic developmental pathway at a stage before protamine transcripts would normally be translated. Rathke and colleagues showed that protamines are incorporated into the spermatid chromatin after the onset of transition protein incorporation (Rathke, 2007), so it is possible that a lack of transition protein causes a developmental block in GLD2 knockdown testes and that the lack of protamine translation in these testes reflects this block. There is evidence that protamine transcripts are translationally repressed for a few days after their transcription and that this repression is dependent upon elements in their 5'UTR (Raja, 2006). Thus, GLD2 could be responsible for controlling the translation of a crucial element that causes the relief of repression at the protamine 5?UTR, while not affecting the poly(A) tail status of the protamine transcript itself (Sartain, 2011).

A third defect in GLD2-deficient spermatogenesis occurs at individualization: actin cones are never detected around late-stage nuclei and the spermatids do not separate from one another. dynamin transcripts are missing in GLD2 knockdown testes. The absence of Dynamin could account for the lack of actin cones at individualization stage nuclei: previous studies have demonstrated that Dynamin is present throughout the actin cones and that disruption of Dynamin function in temperature-sensitive mutants contributes to their instability. Lack of dynamin mRNA could indicate that it is a GLD2 target: lack of dynamin polyadenylation by GLD2 could leave the transcript vulnerable to exonucleases in the cytoplasm, resulting in its degradation. Alternatively, it is possible that the absence of Dynamin in the GLD2 knockdown results from a developmental block during late spermatogenesis, at a time before dynamin RNA would be present (Sartain, 2011).

GLD2 localization might help to identify its target transcripts. Immunofluorescence staining experiments showed that in addition to cytoplasmic localization in spermatocytes, GLD2 localizes to the distal ends of elongated spermatogenic cysts. This is where the polarized growth of the cyst occurs in accordance with axoneme extension; additionally, a group of mRNAs that are transcribed post-meiotically have been shown to localize to the distal end of the spermatogenic cyst (Barreau, 2008). Interestingly, one of these late-transcribed genes is orb, which encodes the Drosophila ortholog of CPEB, the protein necessary for cytoplasmic polyadenylation in Xenopus. The presence of both the CPEB ortholog ORB and the cytoplasmic PAP GLD2 at the end of the cyst where growth is occurring might indicate an involvement of GLD2 in late spermatocyst growth. Taken together, these data suggest that the distal end of the cyst might be a major production center for cyst growth, with the necessary mRNAs regulated post-transcriptionally through cytoplasmic polyadenylation (Sartain, 2011).

This study has shown that GLD2 plays an essential for male, but not female, gametogenesis. This is a unique finding among the literature describing other Gld-2 homologs, where Gld-2 proteins are necessary for some aspect of oogenesis and egg maturation in Drosophila, Xenopus and mice (Barnard, 2004; Benoit, 2008; Cui, 2008) and for the proliferative stages of gametogenesis in hermaphrodite worms (Kadyk, 1998). Drosophila GLD2 plays a role in the male, but not female, germline, and is required in spermatid morphogenesis rather than in proliferative stages. The evidence that Gld2 was retrotransposed to the third chromosome from a duplication of the wisp locus on the X chromosome might give insight to how this unique role for a Gld-2 homolog came about. The phenomenon of meiotic sex chromosome inactivation (MSCI) might have contributed to duplication of the wisp gene and to subsequent retention of Gld2. During spermatogenesis in Drosophila and other animals, the X chromosome is transcriptionally silenced prior to autosomal silencing. Therefore, genes located on the X chromosome have a limited capacity to encode proteins involved in spermatogenesis. Interestingly, an excess of genes has been retrotransposed from the X to the autosomes, and the autosome-derived copies are hypothesized to allow for the escape from X-inactivation. The testis-biased expression and spermatogenic functions of Gld2 suggest that it was selectively retained because it performs a function unavailable to wisp because of MSCI (Sartain, 2011).

It is interesting that Gld2 is crucial for post-meiotic spermatogenesis in Drosophila, whereas all Gld-2 family members analyzed so far in Drosophila and other species play roles specifically at meiosis. It is hypothesized that the function of GLD2 in the male germline reflects its evolutionary origin: duplication of the X-linked wisp locus allows for an autosomal copy that can be expressed in the testis during MSCI. Although this is the first example of a Gld-2 family member with its gametogenic role solely in spermatogenesis, other species might have developed similar mechanisms of translational control in the testes; for example, spermatogenesis in mice is regulated, in part, by a cytoplasmic PAP outside of the Gld-2 family called TPAP (PAPOLB - Mouse Genome Informatics). Further investigation and identification of GLD2 targets in Drosophila testes will help to elucidate how spermatogenesis can be regulated through cytoplasmic polyadenylation (Sartain, 2011).

GLD2 poly(A) polymerase is required for long-term memory

The formation of long-term memory is believed to require translational control of localized mRNAs. In mammals, dendritic mRNAs are maintained in a repressed state and are activated upon repetitive stimulation. Several regulatory proteins required for translational control in early development are thought to be required for memory formation, suggesting similar molecular mechanisms. This study identified the enzyme responsible for poly(A) elongation in the brain and demonstrated that its activity is required specifically for long-term memory. These findings provide strong evidence that cytoplasmic polyadenylation is critical for memory formation, and that GLD2 is the enzyme responsible (Kwak, 2008).

The well characterized molecular mechanisms used in early development to regulate mRNAs are widely thought to be redeployed in somatic cells, including neurons. Localization of mRNA translation to active synapses involves several of the factors used in embryos and may provide the synaptic specificity that is required for memory formation. At present, 6 different mechanisms for translational activation have been proposed (Sutton, 2006; Kindler, 2005). However, very few experiments examine the involvement of these mechanisms in a behaving animal. This report provides evidence that an enzyme known to be critical in the regulation of mRNAs in the germ line also has a role in regulation at adult synapses in vivo. The data support a model in which activity-regulated polyadenylation of synaptic mRNAs is required for long-term memory formation (Kwak, 2008).

The biochemistry and cell biology of DmGLD2 and its protein partners provide evidence for its role in synaptic regulation. DmGLD2 protein is cytoplasmic, interacts physically with eIF4E and dFMR1 proteins, and co-localizes in neurites to mRNPs that contain dFMR1, Pumilio, and other mRNA regulatory proteins (Barbee, 2006). eIF4E, dFMR1, and Pumilio proteins function in the transport and translational control of mRNAs exported from the cell body. The mRNP puncta in neuronal processes are granules in which repressed mRNAs reside during transport. The presence of DmGLD2 in these repressive particles suggests that DmGLD2 may be regulated, perhaps through posttranslational modification or autoregulation of its own mRNA (Rouhana, 2007; Kwak, 2008 and references therein).

GLD2 is likely to be a pivot point in the regulation of dendritic mRNAs. Parallels between cytoplasmic polyadenylation in oocytes and synapses are striking: signals in the 3'UTR are interchangeable and recognized by the same protein, CPEB, that is activated in both cell types through phosphorylation by Aurora kinase. In the germ line, the consummation of the activation process is relief of repression by maskin or 4E-T protein and polyadenylation by the GLD2 PAP (Barnard, 2004). Neuronal CPEB in Drosophila recently has been shown to play a role in long-term memory formation. Mutant flies carrying partial deletion of Orb2, a CPEB homolog expressed specifically in the brain, are defective in forming long-term memory. This study proposes that the PAP GLD-2 is an ultimate target of the Aurora kinase/Maskin/CPEB regulatory pathway and is acutely required during the period of long-term memory formation. As in oocytes, GLD2 probably functions with CPEB in controlling cytoplasmic polyadenylation at synapses; indeed, preliminary results indicate that DmGLD2 and Orb2 co-immunoprecipitate (Kwak, 2008).

This work relies on a DN, catalytically inactive allele of DmGLD2. Only the catalytically inactive allele perturbs memory, demonstrating the specificity of its effects on memory. The simplest interpretation of these findings is that disruption of memory requires titrated components to be sequestered in catalytically inactive complexes. Analysis of analogous mutations in the endogenous GLD2 locus will be instructive (Kwak, 2008).

Despite clear data that polyadenylation occurs at synapses, before this work compelling evidence had not been presented testing its role in memory in an animal. Molecular interpretation of the effects of CPEB disruptions are complicated by CPEB's multiple roles in repression, activation, polyadenylation, and mRNA localization. In the mammalian brain, GLD2 mRNA co-localizes with Pumilio and CPEB mRNAs, suggesting that GLD2 and its partners may all participate in similar neuronal events across species. Indeed, both CPEB/Orb2 and Pumilio are required for in memory formation (Kwak, 2008).

The demonstration that the GLD2 enzyme has a role in long-term memory provides new opportunities. The RNA substrates of GLD2-mediated activation in memory formation are likely to be mRNAs; their identification and validation is a critical next step, as is the use of loss-of-function mutants and perturbations of activity in specific regions of the brain. Effectors that modulate GLD2 activity are predicted to affect memory formation and so have potential clinical value. Elucidation of the molecular steps that regulate translation and of the ties between discrete molecular events and behavior is a major objective. The discovery of GLD2's role in memory will help achieve that goal and will bring studies of GLD2 regulation in the germ line to bear on that elusive mental process (Kwak, 2008).

Wispy, the Drosophila homolog of GLD-2, is required during oogenesis and egg activation

Egg activation is the process that modifies mature, arrested oocytes so that embryo development can proceed. One key aspect of egg activation is the cytoplasmic polyadenylation of certain maternal mRNAs to permit or enhance their translation. wispy (wisp) maternal-effect mutations in Drosophila block development during the egg-to-embryo transition. The wisp gene encodes a member of the GLD-2 family of cytoplasmic poly(A) polymerases (PAPs). The WISP protein is required for poly(A) tail elongation of bicoid, Toll, and torso mRNAs upon egg activation. In Drosophila, WISP and Smaug (SMG) have previously been reported to be required to trigger the destabilization of maternal mRNAs during egg activation. SMG is the major regulator of this activity. SMG is still translated in activated eggs from wisp mutant mothers, indicating that WISP does not regulate mRNA stability by controlling the translation of smg mRNA. The very early developmental arrest associated with wisp mutations was analyzed in detail. Pronuclear migration does not occur in activated eggs laid by wisp mutant females. Finally, WISP function was found to be needed during oogenesis to regulate the poly(A) tail length of dmos during oocyte maturation and to maintain a high level of active (phospho-) mitogen-activated protein kinases (MAPKs) (Cui, 2008).

Although the sperm nucleus appears capable of remodeling and forming a male pronucleus in the absence of WISP function, the sperm asters fail to grow and pronuclear migration does not occur. All the nuclei condense their chromosomes and become associated with a spindle structure, but then arrest at this point. These data are consistent with wisp being essential for some aspect of the tubulin cytoskeleton in oocytes and embryos. WISP also has functions during oogenesis because levels of active (phospho-) MAPKs are severely reduced in wisp oocytes and dmos mRNA poly(A) tails fail to extend in wisp oocytes during oocyte maturation (Cui, 2008).

Given its molecular identity, it is likely that WISP affects these aspects of egg activation and oogenesis by a single mechanism: determination of the poly(A) tail length of certain maternal mRNAs. It is likely that there are more mRNA targets of WISP in addition to the ones identified in this study. Future identification of these mRNA targets whose poly(A) tail increase during oogenesis or upon egg activation is wisp dependent will help to elucidate whether and how products of these genes play roles in oogenesis and egg activation (Cui, 2008).

The Drosophila wispy gene is required for RNA localization and other microtubule-based events of meiosis and early embryogenesis

RNAs are localized by microtubule-based pathways to both the anterior and posterior poles of the developing Drosophila oocyte. A new gene is described, wispy, required for localization of mRNAs to both poles of the egg. Embryos from wispy mothers arrest development after abnormal oocyte meiosis and failure of pronuclei to fuse. Analysis of spindle and chromosome movements during meiosis reveals defects in spindle structures correlated with very high frequencies of chromosome nondisjunction and loss. Spindle defects include abnormally shaped spindles, spindle spurs, and ectopic spindles associated with lost chromosomes, as well as mispositioning of the meiosis II spindles. The polar body nuclei do not associate with their normal monastral arrays of microtubules, the sperm aster is reduced in size, and the centrosomes often dissociate from a mitotic spindle that forms in association with the male pronucleus. wispy is required to recruit or maintain known centrosomal proteins with two types of microtubule organizing centers (MTOCs): (1) the central MTOC that forms between the meiosis II tandem spindles and (2) the centrosomes of the mitotic spindle. It is proposed that the wispy gene product functions directly in several microtubule-based events in meiosis and early embryogenesis (Brent, 2000; full text of article).

Search PubMed for articles about Drosophila wispy

Search PubMed for articles about Drosophila Gld2

Barbee, S. A, et al. (2006). Staufen- and FMRP-containing neuronal RNPs are structurally and functionally related to somatic P bodies. Neuron 52: 997-1009. PubMed ID: 17178403

Bard J., et al. (2000). Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP. Science 289: 1346-1349. PubMed ID: 10958780

Barnard, D. C., et al. (2004). Symplekin and xGLD-2 are required for CPEB-mediated cytoplasmic polyadenylation. Cell 119: 641-651. PubMed ID: 15550246

Barreau C., et al. (2008). Post-meiotic transcription in Drosophila testes. Development 135: 1897-1902. PubMed ID: 18434411

Benoit, P., Papin, C., Kwak, J. E., Wickens, M. and Simonelig, M. (2008). PAP- and GLD-2-type poly(A) polymerases are required sequentially in cytoplasmic polyadenylation and oogenesis in Drosophila. Development 135(11): 1969-79. PubMed ID: 18434412

Brent, A. E., MacQueen, A. and Hazelrigg, T. (2000). The Drosophila wispy gene is required for RNA localization and other microtubule-based events of meiosis and early embryogenesis. Genetics 154: 1649-1662. PubMed ID: 10747060

Chicoine, J., Benoit, P., Gamberi, C., Paliouras, M., Simonelig, M. and Lasko, P. (2007). Bicaudal-C recruits CCR4-NOT deadenylase to target mRNAs and regulates oogenesis, cytoskeletal organization, and its own expression. Dev. Cell 13: 691-704. PubMed ID: 17981137

Cui, J., Sackton, K. L., Horner, V. L., Kumar, K. E. and Wolfner, M. F. (2008). Wispy, the Drosophila homolog of GLD-2, is required during oogenesis and egg activation. Genetics 178(4): 2017-29. PubMed ID: 18430932

Hempel, L. U., et al. (2006). In Drosophila, don juan and don juan like encode proteins of the spermatid nucleus and the flagellum and both are regulated at the transcriptional level by the TAF II80 cannonball while translational repression is achieved by distinct elements. Dev. Dyn. 235(4): 1053-64. PubMed ID: 16477641

Huynh, J. R. and St Johnston, D. (2000). The role of BicD, Egl, Orb and the microtubules in the restriction of meiosis to the Drosophila oocyte. Development 127(13): 2785-94. PubMed ID: 10851125

Kadyk, L. C., and Kimble J. (1998). Genetic regulation of entry into meiosis in Caenorhabditis elegans. Development 125: 1803-1813. PubMed ID: 9550713

Kim, J. H. and Richter, J. D. (2006). Opposing polymerase-deadenylase activities regulate cytoplasmic polyadenylation. Mol. Cell 24: 173-183. PubMed ID: 17052452

Kim J. H. and Richter J. D. (2007). RINGO/cdk1 and CPEB mediate poly(A) tail stabilization and translational regulation by ePAB. Genes Dev. 21: 2571-2579. PubMed ID: 17938241

Kindler, S., et al. (2005). RNA transport and local control of translation. Annu. Rev. Cell Dev. Biol. 21: 223-245. PubMed ID: 16212494

Kwak, J. E., Drier, E., Barbee, S. A., Ramaswami, M., Yin, J. C. and Wickens, M. (2008). GLD2 poly(A) polymerase is required for long-term memory. Proc. Natl. Acad. Sci. 105(38): 14644-9. PubMed ID: 18780789

Lee, M., Choi, Y., Kim, K., Jin, H., Lim, J., Nguyen, T. A., Yang, J., Jeong, M., Giraldez, A. J., Yang, H., Patel, D. J. and Kim, V. N. (2014). Adenylation of maternally inherited microRNAs by Wispy. Mol Cell 56: 696-707. PubMed ID: 25454948

Martin G., Keller W. and Doublie S. (2000). Crystal structure of mammalian poly(A) polymerase in complex with an analog of ATP. EMBO J. 19: 4193-4203. PubMed ID: 10944102

Nakanishi, T., Kubota, H., Ishibashi, N., Kumagai, S., Watanabe, H., Yamashita, M., Kashiwabara, S., Miyado, K. and Baba, T. (2006). Possible role of mouse poly(A) polymerase mGLD-2 during oocyte maturation. Dev. Biol. 289: 115-126. PubMed ID: 16325797

Nakanishi, T., Kumagai, S., Kimura, M., Watanabe, H., Sakurai, T., Kashiwabara, S. and Baba, T. (2007). Disruption of mouse poly(A) polymerase mGLD-2 does not alter polyadenylation status in oocytes and somatic cells. Biochem. Biophys. Res. Commun. 364: 14-19. PubMed ID: 17927953

Pesin, J. A. and Orr-Weaver, T. L. (2007). Developmental role and regulation of cortex, a meiosis-specific anaphase-promoting complex/cyclosome activator. PLoS Genet. 3: e202. PubMed ID: 18020708

Rathke C., et al. (2007). Transition from a nucleosome-based to a protamine-based chromatin configuration during spermiogenesis in Drosophila. J. Cell Sci. 120: 1689-1700. PubMed ID: 17452629

Rouhana, L., Wang, L., Buter, N., Kwak, J. E., Schiltz, C. A., Gonzalez, T., Kelley, A. E., Landry, C. F. and Wickens, M. (2005). Vertebrate GLD2 poly(A) polymerases in the germline and the brain. RNA 11: 1117-1130. PubMed ID: 15987818

Rouhana, L. and Wickens, M. (2007). Autoregulation of GLD-2 cytoplasmic poly(A) polymerase. RNA 13: 188-199. PubMed ID: 17164476

Sartain, C. V., Cui, J., Meisel, R. P. and Wolfner, M. F. (2011). The poly(A) polymerase GLD2 is required for spermatogenesis in Drosophila melanogaster. Development 138(8): 1619-29. PubMed ID: 21427144

Suh, N., Jedamzik, B., Eckmann, C. R., Wickens, M. and Kimble, J. (2006). The GLD-2 poly(A) polymerase activates gld-1 mRNA in the Caenorhabditis elegans germ line. Proc. Natl. Acad. Sci. 103: 15108-15112. PubMed ID: 17012378

Sutton, M. A. and Schuman, E. M. (2006). Dendritic protein synthesis, synaptic plasticity, and memory. Cell 127: 49-58. PubMed ID: 17018276

Vardy, L. and Orr-Weaver, T. L. (2007). The Drosophila PNG kinase complex regulates the translation of cyclin B. Dev. Cell 12: 157-166. PubMed ID: 17199048

Vibranovski M. D., et al. (2010). Direct evidence for postmeiotic transcription during Drosophila melanogaster spermatogenesis. Genetics 186: 431-433. PubMed ID: 20610406

Wang L. T., et al. (2002). A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316. PubMed ID: 12239571

Zhang, Y., et al. (2007). Constraint and turnover in sex-biased gene expression in the genus Drosophila. Nature 450: 233-237. PubMed ID: 17994089

date revised: 2 January 2023

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