InteractiveFly: GeneBrief

hiiragi : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

The hiiragi (hrg) gene encodes a poly(A) polymerase (PAP), an enzyme that attaches adenylyl residues to the 3' untranslated region of mRNAs. hiiragi plays a key role in the development of the wing margin in Drosophila melanogaster. Mutation in hrg is associated with a notched wing phenotype. The levels of expression of wingless and cut at the presumptive wing margins are reduced in the late third-instar larvae of hrg mutants. These results suggest that the product of hrg is required for the normal expression of a series of genes in this region. These results also provide the first evidence that a PAP in Drosophila plays a key role in the early development of the wing margin, acting to regulate the specific expression of a series of genes via, perhaps, control of the processing of the 3' ends of transcripts (Murata, 2001).

The single Drosophila PAP is active in specific polyadenylation in vitro and is involved in both nuclear and cytoplasmic polyadenylation in vivo (Juge, 2002). Therefore, the same PAP can be responsible for both processes. In addition, in vivo overexpression of PAP during embryogenesis does not affect poly(A) tail length during nuclear polyadenylation, but leads to a dramatic elongation of poly(A) tails and a loss of specificity during cytoplasmic polyadenylation, resulting in embryonic lethality. Thus regulation of the PAP level is essential for controlled cytoplasmic polyadenylation and early development. hrg is also probably essential to cell viability since strong hrg mutant germline clones do not survive. The PAP encoded by this gene is involved in both nuclear polyadenylation of rp49 and string of pearls (sop) mRNAs in somatic tissues and cytoplasmic polyadenylation of oskar mRNA in oocytes. This indicates that although the reactions of nuclear and cytoplasmic polyadenylation are not identical, a single PAP is responsible for both in Drosophila (Juge, 2002).

Early steps of development in many species rely on maternally inherited mRNAs because transcription is quiescent at these stages. Therefore, changes in protein synthesis that control early developmental events depend on translational control. One way to regulate translation is by changing the poly(A) tail length of mRNAs in the cytoplasm. Shortening of poly(A) tails correlates with translational repression, whereas lengthening of poly(A) tails induces translation. In Drosophila embryos, cytoplasmic polyadenylation is crucial for initiation of development; it activates translation of several molecules essential for axis formation, such as the anterior morphogen Bicoid (Salles, 1994), Hunchback (Wreden, 1997) and Toll (Schisa, 1998). Cytoplasmic polyadenylation has also been proposed to regulate translation of the posterior determinant Oskar (Chang, 1999) during Drosophila oogenesis (Juge, 2002).

The molecular mechanism of cytoplasmic polyadenylation has been analysed extensively in Xenopus oocytes, and some aspects of the reaction are similar to that of nuclear polyadenylation. Nuclear polyadenylation consists of endonucleolytic cleavage of pre-mRNAs followed by the synthesis of a poly(A) tail onto the upstream cleavage product (reviewed by Zhao, 1999). Poly(A) addition can be reconstituted in vitro from three purified mammalian factors: poly(A) polymerase (PAP), cleavage and polyadenylation specificity factor (CPSF) and poly(A)-binding protein II [PABP2, the nuclear poly(A)-binding protein]. CPSF is a complex of four proteins that binds the polyadenylation signal AAUAAA located upstream of the cleavage site. Recognition of the poly(A) site also requires cleavage stimulation factor (CstF) that binds to a GU/U-rich element downstream of the cleavage site and interacts with CPSF. PAP catalyses the polyadenylation reaction, but is also required for efficient cleavage of pre-mRNAs in vitro (Christofori, 1989; Takagaki, 1989). PAP by itself does not recognize pre-mRNAs specifically. Specificity requires the AAUAAA element and CPSF that binds PAP through its 160 kDa subunit (Murthy, 1995). Even in the presence of CPSF, PAP activity remains weak; it is again stimulated by binding of PABP2 to the poly(A) tail (Wahle, 1991b). Together, CPSF and PABP2 stimulate PAP activity by holding PAP on the RNA such that a full-length poly(A) tail is synthesized in a single processive event (Bienroth, 1993). When the poly(A) tail has reached its complete length, elongation is no longer processive and becomes slow and distributive. PABP2 is required (Wahle, 1995) for this poly(A) tail length control (Juge, 2002).

Cytoplasmic polyadenylation in Xenopus relies on two sequences: the nuclear polyadenylation signal, AAUAAA, and an upstream U-rich element called the cytoplasmic polyadenylation element (CPE). CPE-dependent polyadenylation can be recapitulated in vitro in the presence of purified bovine CPSF and PAP (Bilger, 1994), indicating a role for CPSF. Indeed, a cytoplasmic form of CPSF has been identified in Xenopus oocytes (Dickson, 1999). CPEs are bound by CPEB (Drosophila homolog: Orb), a major component of the reaction (Hake, 1994; Mendez, 2001). Cytoplasmic polyadenylation during Xenopus oocyte maturation is triggered by phosphorylation of CPEB, which stimulates a direct interaction between CPEB and the 160 kDa subunit of CPSF (Mendez, 2000). Thus, the role of CPEB during cytoplasmic polyadenylation would be to recruit CPSF into an active polyadenylation complex containing a PAP (Juge, 2002).

In Drosophila, the role of CPEs has not been addressed, and the polyadenylation signal is dispensable in some cases, since embryonic cytoplasmic polyadenylation occurs on a bicoid engineered mRNA deleted for this element (Salles, 1994). Although genes encoding the four subunits of CPSF are present in the Drosophila genome (Mount, 2000), their role in cytoplasmic polyadenylation has not been determined. The Drosophila homolog of CPEB is the Orb protein. orb encodes germline-specific proteins different in male and female, and its function has been determined in the female germline. Strong orb mutants arrest oogenesis early, before the formation of the 16-cell cyst that would normally differentiate into nurse cells and one oocyte. Using a weaker allele, orb^mel, Orb was shown to be required for anchoring of oskar mRNA at the posterior pole of the oocyte. However, this could result from a failure in oskar mRNA translation since Oskar protein is required for anchoring its own mRNA at the posterior pole. A recent study (Chang, 1999) suggests that Orb could have a function analogous to that of CPEB in cytoplasmic polyadenylation. In orb mutant egg chambers, the level of Oskar protein is decreased and poly(A) tails of oskar mRNAs are shortened (Juge, 2002).

Another key component required in a functional cytoplasmic polyadenylation complex is a PAP. In vertebrates, multiple PAP isoforms have been identified. Initially, two PAP isoforms were described, PAP I (70 kDa) and PAP II (83 kDa), that differ in their C-terminus (Raabe, 1991; Wahle, 1991a). Analysis of PAP mRNAs in mouse revealed that these two PAP isoforms are generated by alternative splicing (Zhao, 1996). Truncated forms of PAP RNAs corresponding to the 5' half of the gene have also been identified in several species (Wahle, 1991a; Ballantyne, 1995; Gebauer, 1995; Zhao, 1996). However, these truncated RNAs are thought not to be translated in vivo, and the corresponding proteins produced in baculovirus or in Escherichia coli are inactive in vitro (Wahle, 1991a; Martin, 1996; Zhao, 1996). In addition to the PAP gene, two new PAP-encoding genes have been identified recently in mammals, neo-PAP (or PAPg) and TPAP. The neo-PAP gene encodes a single protein that shows 60% identity to human PAP II and has identical properties as those of PAP II in in vitro assays (Kyriakopoulou, 2001; Perumal, 2001; Topalian, 2001). TPAP is encoded by an intronless gene (Kashiwabara, 2000; Lee, 2000). Interestingly, TPAP is expressed specifically in testis, and the protein is specifically cytoplasmic in spermatogenic cells where cytoplasmic polyadenylation is active. TPAP was therefore proposed to be responsible for cytoplasmic polyadenylation in mouse testis. Although the function of these four PAP isoforms has not been investigated in vivo, it seems plausible that they have specific functions. Results on TPAP suggest that different PAPs are responsible for nuclear and cytoplasmic polyadenylation in vertebrates (Juge, 2002).

To address a possible role for hrg in cytoplasmic polyadenylation during early development, germline clones homozygous for hrg^PAP45, hrg^PAP21 or hrg^PAP12 were induced. No germline clones were obtained for any of these mutants, possibly as a result of a requirement of PAP for cell viability. Therefore genetic interactions were studied between hrg and orb, which is known to be involved in cytoplasmic polyadenylation. Females homozygous for the weak orb^mel allele produce egg chambers at all stages and lay eggs, 30% of which show a ventralized phenotype. hrg lethal mutants act as dominant enhancers of the orb^mel phenotype, since hrg^PAP45/+; orb^mel and hrg^PAP21/+; orb^mel females lay almost no eggs. In these females, oogenesis stops most frequently at stage 7/8, after which egg chambers degenerate, even though one or two stage 14 oocytes per ovary can be observed. Poly(A) tails of oskar mRNA are shortened in orb mutant ovaries (Chang, 1999). The defect of these poly(A) tails were analyzed in hrg^- /+; orb^mel mutants by PAT assays. oskar mRNA poly(A) tails were measured to be up to 135 residues in wild-type ovaries. These poly(A) tails are weakly reduced in orb^mel, but severely reduced in hrg^- /+;orb^mel double mutant ovaries, their maximal length reaching 40- 50 residues. These short poly(A) tails do not result from the oogenesis defect in hrg^- /+; orb^mel females, since unrelated mutants that stop oogenesis early show wild-type poly(A) tails of oskar mRNA (Chang, 1999). These poly(A) tails were also found to be of wild-type length in hrg^PAP21/+ and hrg^PAP45/+ ovaries. This shows that the strong shortening of oskar poly(A) tails in hrg^- /+; orb^mel mutants does not result from an additive effect of two phenotypes, but from a synergistic effect of the two mutants due to a simultaneous decrease in PAP and Orb protein levels. This strongly suggests that hrg and orb are involved together in cytoplasmic polyadenylation. This was confirmed by measurements of poly(A) tails of a control mRNA, sop, which is thought not to be regulated by cytoplasmic polyadenylation. Poly(A) tails of sop mRNAs are unaffected in orb^mel as well as in hrg^- /+; orb^mel mutant ovaries. It was verified that shortening of oskar mRNA poly(A) tails in hrg^- /+; orb^mel mutants leads to a reduction of Oskar protein level, by immunostaining of ovaries with anti-Oskar. Oskar accumulates at the posterior of the oocyte from stage 9 onwards. The amount of Oskar decreases in orb^mel oocytes. This amount decreases again in hrg^- /+; orb^mel oocytes to a barely detectable level (Juge, 2002).

Taken together, these results show that hrg and orb cooperate in poly(A) tail lengthening during cytoplasmic polyadenylation and that alteration of this process affects protein accumulation and oogenesis (Juge, 2002).

To address whether cytoplasmic polyadenylation could be affected by the level of PAP, PAP was overexpressed in the female germline using the UASp-hrg transgene and nos-Gal4. This overexpression does not cause gross alteration of oogenesis, but is extremely detrimental to embryogenesis, leading to 99% lethality of the progeny. These embryos stop development early, before cleavage of nuclei. Cytoplasmic polyadenylation was analysed in these embryos by measuring poly(A) tails of bicoid mRNA that is regulated by this process during embryogenesis (Salles, 1994). In the wild-type, poly(A) tails of bicoid mRNA lengthen from 80 residues in oocytes to 170 residues in 1 h embryos. This elongation of the poly(A) tails induces Bicoid protein synthesis in early embryos (Salles, 1994). Following overexpression of PAP, poly(A) tails of bicoid mRNA strongly increase in length, with a pool of mRNAs bearing a 250 residue poly(A) tail in oocytes and most mRNAs having a poly(A) tail between 300 and 600 residues in 1 h embryos. The fact that bicoid mRNA poly(A) tails lengthen in 0- 1 h embryos, at a stage when there is no transcription, shows that the process affected by PAP overexpression is cytoplasmic polyadenylation. This was confirmed by showing that when PAP is overexpressed ubiquitously in somatic cells with the da-Gal4 driver, poly(A) tails of sop mRNA are not affected. Therefore, poly(A) tail length control during nuclear polyadenylation is not altered by PAP overexpression, although the level of somatic overexpression is in the same range as that of germline overexpression. Surprisingly, although sop mRNA does not undergo cytoplasmic polyadenylation in wild-type embryos, overexpression of PAP in the female germline leads to a strong lengthening of sop mRNA poly(A) tails by cytoplasmic polyadenylation. Similar results were found for rp49 mRNA. This indicates that the increasing PAP level affects both poly(A) tail length control and specificity during cytoplasmic polyadenylation. Bicoid protein accumulation and bicoid mRNA poly(A) tail length was correlated by immunostaining of ovaries and embryos with anti-Bicoid. Poly(A) tail elongation of bicoid mRNA in oocytes, following PAP overexpression, does not induce translation since no Bicoid is detected in UASp-hrg; nos-Gal4 oocytes. Therefore, in oocytes, long poly(A) tails are not sufficient to induce bicoid mRNA translation. In embryos where PAP is overexpressed, poly(A) tail lengthening correlates with a precocious accumulation of Bicoid and with an increase in Bicoid protein level. These data demonstrate that a tight regulation of PAP level is essential to control cytoplasmic polyadenylation and to early development (Juge, 2002).

Two important conclusions can be drawn from this work. (1) A single isoform of PAP is able to perform both reactions of nuclear and cytoplasmic polyadenylation in vivo. (2) A controlled level of PAP is essential for specificity of cytoplasmic polyadenylation and for poly(A) tail length control during cytoplasmic polyadenylation (Juge, 2002).

Although hrg produces three mRNAs, they all encode the same protein. Western blots on Drosophila extracts confirm the presence of a single PAP isoform in Drosophila. As expected for a gene responsible for such a fundamental process as polyadenylation, hrg is essential for viability. Strong hrg mutants are lethal at late embryonic and larval stages. hrg is also probably essential to cell viability as strong hrg mutant germline clones do not survive. PAP encoded by this gene is involved in both nuclear polyadenylation of rp49 and sop mRNAs in somatic tissues and cytoplasmic polyadenylation of oskar mRNA in oocytes. This indicates that although the reactions of nuclear and cytoplasmic polyadenylation are not identical, a single PAP is responsible for both in Drosophila (Juge, 2002).

Recently, a new class of cytoplasmic PAPs was discovered that is not related in sequence to conventional PAPs (Wang, 2002). These proteins are widespread in eukaryotes and three homolog s exist in Drosophila. A member in nematodes functions in germline and embryonic development; therefore, this class of proteins was proposed to play a role in cytoplasmic polyadenylation during development, in addition to conventional PAPs (Juge, 2002).

Drosophila PAP indeed has a poly(A) polymerase activity in vitro, in reconstituted specific polyadenylation assays. Stimulation of Drosophila PAP activity by bovine CPSF indicates that Drosophila PAP and bovine CPSF interact. In mammalian PAP, the region thought to be involved in interaction with CPSF overlaps the first NLS (Thuresson, 1994) and this domain is conserved in Drosophila (Juge, 2002).

Drosophila PAP has a role in vivo in nuclear polyadenylation. In hrg mutant larvae, poly(A) tails of rp49 and sop mRNAs are short. These short poly(A) tails probably result from the decay of rp49 and sop transcript pools and a lack of newly polyadenylated mRNAs. In vitro studies led to the general belief that PAP is required for the cleavage of pre-mRNAs at poly(A) sites (Zhao, 1999). However, it is not the case in vivo, at least for two different pre-mRNAs. Overexpression of PAP in somatic tissues does not alter poly(A) tail length of sop mRNAs. This indicates that the amount of PAP is not limiting in vivo for nuclear polyadenylation. This is not unexpected since, during the polyadenylation reaction, PABP2 plays an important role in stimulating PAP. Once PABP2 is bound to the newly synthesized 10 residue poly(A) tail, a single PAP molecule is required to polymerize the complete poly(A) tail (Bienroth, 1993). PABP2 also controls the length of the poly(A) tail, and this function of PABP2 may prevent the recruitment of new PAP molecules in the complex, and the poly(A) tail to lengthen even if more PAP is present (Juge, 2002).

hrg, in conjunction with orb, is involved in cytoplasmic polyadenylation of oskar mRNA during oogenesis. The control of oskar mRNA translation is very complex. oskar mRNA is transported to the posterior pole of the oocyte and translation does not start before this posterior localization. An essential determinant in translational repression during oskar mRNA transport is the Bruno protein. Although the mechanism underlying translational repression by Bruno currently is unknown, it has been shown, in a cell-free system, to be independent of poly(A) tail length. Therefore, although oskar mRNA undergoes cytoplasmic polyadenylation (Chang, 1999), the role of this regulation in the control of oskar mRNA translation is unclear. The data provide further evidence that cytoplasmic polyadenylation has a role in Oskar expression, since, when this process is impaired, Oskar does not accumulate at the posterior pole of the oocyte. Regulation of oskar mRNA poly(A) tail length probably represents an additional level of control of Oskar expression. In hrg^PAP21/+; orb^mel mid-oocytes, the amount of oskar mRNA is low. This suggests that cytoplasmic polyadenylation could be required to unbalance rapid deadenylation and decay of oskar mRNA (Juge, 2002).

Other mRNAs regulated by cytoplasmic polyadenylation in Drosophila oogenesis have not been identified, but many are to be expected. Strong alleles of orb stop oogenesis early, and a recent study indicates that orb is required for oocyte determination. This suggests that Orb regulates translation of mRNAs that have a function very early during oogenesis. In agreement with this, hrg^- /+; orb^mel females stop oogenesis at an earlier stage than orb^mel females. mRNAs regulated by cytoplasmic polyadenylation during early oogenesis have been identified in mouse. They encode two proteins of the synaptonemal complex, a complex required for recombination during meiosis, and these proteins are not produced in CPEB knockout mouse oocytes (Juge, 2002).

A crucial conclusion from these data is that a tightly regulated level of PAP has a major role in cytoplasmic polyadenylation. Overexpressing PAP in the female germline results in a strong elongation of poly(A) tails of bicoid and sop, mRNAs that are and are not regulated by cytoplasmic polyadenylation, respectively. Therefore, increasing the level of PAP alters both poly(A) tail length control and the specificity of cytoplasmic polyadenylation for certain mRNAs. This deregulation leads to early embryonic lethality. That a low level of PAP is important for cytoplasmic polyadenylation regulation correlates with the repression of PAP activity by phosphorylation in Xenopus oocytes during meiotic maturation, when cytoplasmic polyadenylation occurs (Ballantyne, 1995; Colgan, 1998). In contrast, overexpression of PAP in somatic tissues does not affect poly(A) tail length control during nuclear polyadenylation. This difference has mechanistic implications and suggests that if PABP2 is involved at some step in cytoplasmic polyadenylation, as indeed is the case, its role is different from that during nuclear polyadenylation. That an increase of PAP level leads to unregulated very long poly(A) tails suggests that poly(A) tail synthesis during cytoplasmic polyadenylation mainly depends on the ability of PAP to interact with mRNAs, and that cytoplasmic polyadenylation never enters a processive state where a single PAP molecule would be sufficient to complete a poly(A) tail in one event. This correlates with the slowness of the reaction during embryogenesis where elongation of bicoid mRNA poly(A) tail (Salles, 1994) extends for 1- 1.5 h of development (Juge, 2002).

In Xenopus oocytes, CPEB makes the cytoplasmic polyadenylation reaction specific to some mRNAs, and it is probable that Orb has the same role in Drosophila. The loss of specificity of the reaction following PAP overexpression indicates that the PAP level also has an active role in this specificity. In this context, cytoplasmic poly(A) tail elongation of sop and other mRNAs that do not normally undergo this reaction probably requires neither Orb, nor another protein that would recognize these mRNAs specifically. This suggests that an active cytoplasmic polyadenylation complex can form in the absence of Orb/CPEB, that would contain CPSF and PAP only. In vitro studies have also led to this conclusion (Dickson, 2001). However, whether or not such a complex is actually responsible for cytoplasmic polyadenylation of some mRNAs in vivo under normal conditions, and in that case what makes the reaction specific, represent a challenge for further studies (Juge, 2002).

REGULATION

Measurement of PAP activity

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 hrg^PAP12 first instar larvae and controlled by an RT- PCR with primers located in the coding region of pgk, another gene expressed ubiquitously. In hrg^PAP12, 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 hrg^PAP45 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 hrg^PAP12 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).

Protein Interactions

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).

PAP- and GLD-2-type poly(A) polymerases are required sequentially in cytoplasmic polyadenylation and oogenesis in Drosophila.

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). 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, 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).

DEVELOPMENTAL BIOLOGY

The expression profile of PAP during Drosophila development was assayed using a polyclonal antibody. During oogenesis, PAP is detected in both the nucleus and cytoplasm of nurse cells and follicle cells. PAP is also present at a low level in oocyte nucleus and cytoplasm. PAP was overexpressed in the female germline using a UASp-hrg transgene under the control of the female germline-specific driver nanos-Gal4:VP16 (nos- Gal4). In UASp-hrg; nos-Gal4 females, PAP accumulates to a high level in nuclei of nurse cells and oocyte and to a lesser extent in oocyte cytoplasm. Maternally provided PAP is detected in just laid embryos where the protein is distributed uniformly. During embryogenesis, the amount of PAP increases until cellularized blastoderm stage and remains stable during gastrulation. The subcellular distribution of PAP was analysed in cellularized blastoderm embryos. PAP accumulates in nuclei and is present at a lower level in the cytoplasm, as was reported for PAP II in human somatic cells (Schul, 1998; Kyriakopoulou, 2001). A high level of PAP accumulates in early embryos coming from females where PAP is overexpressed in the germline. In contrast, PAP is not detected in hrg^PAP12 mutant embryos that show no hrg early zygotic transcription. This shows that the antibody is specific for PAP. A major protein is detected in Drosophila extracts with this antibody by Western blot. This protein has a mol. wt of 75 kDa, which is the expected molecular weight for Drosophila PAP. Its level increases during the first hour of embryogenesis; it is very abundant in embryos from females overexpressing PAP in the germline and in late embryos overexpressing PAP ubiquitously, and is absent in hrg^PAP12 mutant larvae (Juge, 2002).

These data show that hrg encodes a single form of PAP. This protein is mostly nuclear in somatic cells and is present in the cytoplasm of oocytes and early embryos where cytoplasmic polyadenylation takes place (Juge, 2002).

EFFECTS OF MUTATION

Since mutation at the hrg locus results in loss of the normal wing margin, an examination was carried out to see whether expression of the ct and wg genes might be altered by mutations in the hrg gene, namely, by hrg^P1 and hrg¹⁰ (Murata, 1996b). Reduced expression of lacZ directed by the ct margin enhancer along the boundary of the dorsal/ventral (D/V) compartment was evident around the anterior/posterior (A/P) boundary and, for the most part, in both the anterior and posterior regions of the wing pouch. Reduced expression of wg was observed specifically at the wing margin, while expression of wg in other regions of the wing disc, as well as in other imaginal discs such as the leg discs, was unaffected by the mutations in the hrg gene. Thus, it appears that hrg might act specifically in the regulation of expression of the wg gene at the presumptive wing margin rather than in the regulation of the overall expression of wg. The reduced levels of expression of ct and wg that are associated with mutations in the hrg gene suggest that loss of expression of these genes might be responsible for the loss of the normal wing-margin structure in the hrg mutants (Murata, 2001).

Since the duplication Dp(2;Y)53D;57C, which covers the hrg locus (Murata, 1996), rescues the notched phenotype of adult wings of hrg^P1 mutant flies, fragments of genomic DNA were sought that encode genes that could restore hrg activity. Among the DNA fragments isolated from a wild-type genomic DNA library, a 6.8-kb SalI fragment (hrg⁷²) was identified that was able to rescue the adult wing phenotype when introduced into hrg^P1 and hrg¹⁰ mutants by P element-mediated transformation. DNA sequence analysis of phrg⁷² and embryonic cDNA clones identified the hrg^E transcript (embryonic hrg) as a transcript that corresponds to part of the genomic DNA of phrg⁷². Northern blots of larval RNA with a cDNA probe for hrg^E and the phrg⁷² probe demonstrated that a 4.0-kb transcript is the major hybridizing species in wild-type larvae and that the level of this transcript is eightfold less in hrg^P1 and threefold less in hrg¹⁰ mutants, respectively. Neither hrg^P1 nor hrg¹⁰ are null alleles of the hrg gene because RT-PCR detects coding regions of hrg in these alleles (Murata, 2001).

Using the Gal4 line C-765, it was shown that introduction of the UAS-hrgE transgene rescues the wing phenotype in hrg¹⁰ mutant flies. Thus, hrg^E is sufficient to reverse the hrg mutation. Taken together, the results suggest that the level of expression of hrg is reduced in flies with the mutant alleles of the hrg gene and that the reduced expression of hrg results in failure to develop a normal wing (Murata, 2001).

The hrg gene appears to function at the wing margin both correctly and specifically since no defects other than abnormal wing margins are found in hrg^P1 and hrg¹⁰ flies (Murata, 1996). The expression of hrg is detected in most tissues, including the imaginal discs, fat bodies, salivary glands, and muscles of wild-type larvae. Such ubiquitous expression of hrg is impaired in larvae with mutant hrg alleles (Murata, 2001).

To examine whether a defect in PAP might be responsible for the wing phenotype of hrg mutants, cDNA for bovine PAPII was introduced into flies by P element-mediated transformation. All hrg¹⁰ flies with the Gal4 line MS1096 and UAS-bPAPII had wild-type wings. The wing phenotype of the hrg¹⁰ mutants did not return to the wild-type wing phenotype when hrg^E that encodes not aspartic acid, but alanine at position 175, was expressed in the hrg¹⁰ mutants by introduction of UAS-hrgD175A and the C-765 Gal4 line. These results suggest that the wing phenotype of hrg might be due to reduction of the expression of a gene that encodes a protein that is functionally similar to PAP (Murata, 2001).

The hrg gene was localized by in situ hybridization to position 56E5- 6 on chromosome II. A collection of 21 P{lacW}-induced lethal mutants on chromosome II, containing the P insertion in the vicinity of region 56E5- 6, were screened by Southern hybridization. In three of these stocks, l(2)k07618, l(2)k07609 and l(2)k07626, the P element was found to be inserted in the 5'-untranslated region (UTR) of hrg. DNA from these three stocks shows the same restriction profile, suggesting that it contains the same P-element insertion. The insertion was mapped in the l(2)k07618 stock and is located 261 bp downstream of the hrg 5'-most transcription start site. Although this insertion was isolated in a P-induced lethal mutant collection, it does not cause lethality. The mutation inducing lethality in the l(2)k07618 stock was removed by recombination. This stock was named hrg^PAP2. hrg^PAP2 is viable and fertile, although ~50% of homozygous hrg^PAP2 individuals die as first instar larvae. New hrg mutants were generated by imprecise excision of the P-element in hrg^PAP2. Three homozygous lethal mutants, hrg^PAP45, hrg^PAP21 and hrg^PAP12, that show a deletion in the coding sequence were used in further studies. The first six residues and the first 44 residues are deleted in hrg^PAP45 and in hrg^PAP21, respectively. In hrg^PAP12, more than the N-terminal third of PAP (243 residues), including the catalytic core, is deleted. In all three mutants, most of the 5'-UTR of the longest mRNAs as well as the embryonic transcription start site are missing. The three mutants are lethal from late embryonic to second instar larval stages, with hrg^PAP45 showing the weakest phenotype and hrg^PAP12 the strongest. They do not complement each other and are, therefore, alleles of the same gene. Late embryos of all three mutants show no strong phenotype; however, they present a slight defect in head skeleton (distortion of the dorsal bridge). Lethality of hrg^PAP45 and hrg^PAP21 is rescued with a hrg genomic transgene. However, only 20% for hrg^PAP45 and 5% for hrg^PAP21 of the expected rescued progeny survive to adulthood, suggesting that hrg in the transgene is not fully expressed. Lethality of the strongest allele hrg^PAP12 is not rescued with the genomic transgene, but is rescued with the transgene UASp-hrg expressed ubiquitously with the driver daughterless-Gal4 (da-Gal4). It as verified that hrg mutants described earlier (Murata, 2001) are alleles of the gene described in this study. hrg^P1 is not lethal but shows a notched wing phenotype. hrg^PAP12 does not complement hrg^P1, since hrg^P1/hg^PAP12 adults have a pronounced notched wing phenotype (Juge, 2002).

Taken together, these data demonstrate that strong alleles of the hrg gene have been induced that encode Drosophila PAP and that the lack of PAP in Drosophila is lethal (Juge, 2002).

EVOLUTIONARY HOMOLOGS

Two conserved regulatory cytoplasmic poly(A) polymerases, GLD-4 and GLD-2, regulate meiotic progression in C. elegans

Translational regulation is heavily employed during developmental processes to control the timely accumulation of proteins independently of gene transcription. In particular, mRNA poly(A) tail metabolism in the cytoplasm is a key determinant for balancing an mRNA's translational output and its decay rate. Noncanonical poly(A) polymerases (PAPs), such as germline development defective-2 (GLD-2), can mediate poly(A) tail extension. Little is known about the regulation and functional complexity of cytoplasmic PAPs. This study report the discovery of C. elegans GLD-4, a cytoplasmic PAP present in P granules that is orthologous to Trf4/5p from budding yeast. GLD-4 enzymatic activity is enhanced by its interaction with GLS-1, a protein associated with the RNA-binding protein GLD-3. GLD-4 is predominantly expressed in germ cells, and its activity is essential for early meiotic progression of male and female gametes in the absence of GLD-2. For commitment into female meiosis, both PAPs converge on at least one common target mRNA—i.e., gld-1 mRNA—and, as a consequence, counteract the repressive action of two PUF proteins and the putative deadenylase CCR-4. Together these findings suggest that two different cytoplasmic PAPs stabilize and translationally activate several meiotic mRNAs to provide a strong fail-safe mechanism for early meiotic progression (Schmid, 2009).

Poly(A) polymerase and early development of Xenopus

Poly(A) can be added to mRNAs both in the nucleus and in the cytoplasm. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs provides a common mechanism of translational control. To begin to understand the regulation of polyadenylation activities during early development, poly (A) polymerases (PAPs) were analyzed in oocytes and early embryos of the frog, Xenopus laevis. A PAP cDNA that corresponds to a maternal mRNA present in frog oocytes was cloned and sequenced. This PAP is similar in size and sequence to mammalian nuclear PAPs. By immunoblotting using monoclonal antibodies raised against human PAP, it has been demonstrated that oocytes contain multiple forms of PAP that display different electrophoretic mobilities. The oocyte nucleus contains primarily the slower migrating forms of PAP, whereas the cytoplasm contains primarily the faster migrating species. The nuclear forms of PAP are phosphorylated, accounting for their retarded mobility. During oocyte maturation and early postfertilization development, preexisting PAPs undergo regulated phosphorylation and dephosphorylation events. Using the cloned PAP cDNA, it has been demonstrated that the complex changes in PAP forms seen during oocyte maturation may be due to modifications of a single polypeptide. These results demonstrate that the oocyte contains a cytoplasmic polymerase closely related to the nuclear enzyme and suggest models for how its activity may be regulated during early development (Ballantyne, 1995).

p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevents complete phosphorylation and MPF-mediated repression. PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites is required for repression of PAP activity, and partial phosphorylation has no detectable effect. The consensus sites are phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites are phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remain so throughout maturation, while the non-consensus sites do not become fully phosphorylated until after 12 h of metaphase II arrest. It is proposed that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. The possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle is discussed (Colgan, 1998).

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. The hypothesis that CPSF is required for all polyadenylation reactions is discussed, but the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzes polyadenylation and stimulates translation approximately 40-fold; stimulation is exclusively cis-acting, but is independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation require PAPs catalytic core, but does not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA (Dickson, 2001).

Mammalian poly(A) polymerases

Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, the mouse PAP gene and an intronless pseudogene were isolated from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which here is termed PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive (Zhao, 1996).

Deletion and substitution mutants of bovine poly(A) polymerase have been tested, and a small region has been identified that overlaps with a nuclear localization signal and binds to the RNA primer. Systematic mutagenesis of carboxylic amino acids led to the identification of three aspartates that are essential for catalysis. Sequence and secondary structure comparisons of regions surrounding these aspartates with sequences of other polymerases revealed a significant homology to the palm structure of DNA polymerase beta, terminal deoxynucleotidyltransferase and DNA polymerase IV of Saccharomyces cerevisiae, all members of the family X of polymerases. This homology extends as far as cca: tRNA nucleotidyltransferase and streptomycin adenylyltransferase, an antibiotic resistance factor (Martin, 1996).

A single pre-mRNA could generate multiple forms of mammalian poly(A) polymerase mRNAs by alternative splicing or alternative polyadenylation. A cDNA encoding a testis-specific poly(A) polymerase has been isolated. The transcription level of Papt in testis of a 2 weeks old mouse is much lower than that of the general poly(A) polymerase gene, Pap. However, the transcription ratio of Papt to Pap is reversed in testis of a 4 weeks old mouse. Transient expression analysis showed that GFP-Papt fusion protein is present both in the nucleus and cytoplasm of HeLa cells. These results suggest that Papt is involved in polyadenylation of transcripts expressed during spermatogenesis (Lee, 2000).

cDNA clones have been identified encoding a testis-specific poly(A) polymerase, termed TPAP, a candidate molecule responsible for cytoplasmic polyadenylation of preexisting mRNAs in male haploid germ cells. The TPAP gene is most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contains 642 residues, and shares a high degree of identity (86%) with that of a nuclear poly(A) polymerase, PAP II. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacks an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAP II. Recombinant TPAP produced by an in vitro transcription/translation system is capable of incorporating the AMP moiety from ATP into an oligo(A)₁₂ RNA primer in the presence of MnCl₂. Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognizes a 70-kDa protein in the cytoplasm of spermatogenic cells. These results suggest that TPAP may participate in the additional extension of mRNA poly(A) tails in the cytoplasm of male germ cells, and may play an important role in spermiogenesis, probably through the stabilization of mRNAs (Kashiwabara, 2000).

The 3'-terminal adenylic acid residue in several human small RNAs including signal recognition particle (SRP) RNA, nuclear 7SK RNA, U2 small nuclear RNA, and ribosomal 5S RNA is caused by a post-transcriptional adenylation event. Using the Alu portion of the SRP RNA as a substrate in an in vitro adenylation assay, an adenylating enzyme has been identified that adds adenylic acid residues to SRP/Alu RNA from the HeLa cell nuclear extract. All the peptide sequences obtained by microsequencing of the purified enzyme matched a unique human cDNA corresponding to a new adenylating enzyme having homologies to the well characterized mRNA poly(A) polymerase. The amino terminus region of the human SRP RNA adenylating enzyme showed approximately 75% homology to the amino terminus of the human mRNA poly(A) polymerase that includes the catalytic domain. The carboxyl terminus of the human SRP RNA adenylating enzyme showed less than 25% homology to the carboxyl terminus of poly(A) polymerase, which interacts with other factors and provides specificity. The SRP RNA adenylating enzyme is coded for by a gene located on chromosome 2 in contrast to the poly(A) polymerase gene, which is located on chromosome 14. A recombinant protein for the SRP RNA adenylating enzyme was prepared, and its activity was compared with the purified enzyme from HeLa cells. The data indicate that in addition to the SRP RNA adenylating enzyme, other factors may be required to carry out accurate 3'-end adenylation of SRP RNA (Perumal, 2001).

Poly(A) polymerase (PAP) plays an essential role in polyadenylation of mRNA precursors, and it has long been thought that mammalian cells contain only a single PAP gene. A human PAP, called neo-PAP, is encoded by a previously uncharacterized gene. cDNA was isolated from a tumor-derived cDNA library encoding an 82.8-kDa protein bearing 71% overall similarity to human PAP. Strikingly, the organization of the two PAP genes is nearly identical, indicating that they arose from a common ancestor. Neo-PAP and PAP were indistinguishable in in vitro assays of both specific and nonspecific polyadenylation and also endonucleolytic cleavage. Neo-PAP produced by transfection is exclusively nuclear, as demonstrated by immunofluorescence microscopy. However, notable sequence divergence between the C-terminal domains of neo-PAP and PAP suggests that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP does not show evidence of phosphorylation on Western blot analysis; this was unexpected in the context of a conserved cyclin recognition motif and multiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intriguingly, Northern blot analysis demonstrates that each PAP displays distinct mRNA splice variants, and both PAP mRNAs are significantly overexpressed in human cancer cells compared to expression in normal or virally transformed cells. Neo-PAP may therefore be an important RNA processing enzyme that is regulated by a mechanism distinct from that utilized by PAP (Topalian, 2001).

Nuclear distribution and function of poly(A) polymerase

A detailed study was performed of the spatial distribution of a set of mRNA 3' processing factors in human T24 cells. A key enzyme in RNA 3' processing, poly(A) polymerase (PAP), was found in the cytoplasm and throughout the nucleus in a punctated pattern. A subset of the various isoforms of PAP is specifically concentrated at sites of RNA synthesis in the nucleoplasm. Additionally, the other factors necessary for RNA 3' processing, such as CstF, CPSF, and PABII, were also found at these transcription sites. These data show that the set of 3' processing factors that are presumed to be necessary for most RNA 3' cleavage and polyadenylation is indeed found at sites of RNA synthesis in the nucleoplasm. Furthermore, sites of RNA synthesis that are particularly enriched in both PAP and PABII are found at the periphery of irregularly shaped domains, called speckles, which are known to contain high concentrations of splicing factors and poly(A) RNA. Disruption of RNA 3' processing by the drug 9-beta-D-arabinofuranosyladenine causes the speckles to break up into smaller structures. These findings indicate that there is a spatial and structural relationship between 3' processing and the nuclear speckles. These studies reveal a complex and distinct organization of the RNA 3' processing machinery in the mammalian cell nucleus (Schul, 1998).

Poly(A) polymerase (PAP) is present in multiple forms in mammalian cells and tissues. The 90-kDa isoform is the product of the gene PAPOLG, which is distinct from the previously identified genes for poly(A) polymerases. The 90-kDa isoform is referred to as human PAP gamma (hsPAP gamma). hsPAP gamma shares 60% identity to human PAPII (hsPAPII) at the amino acid level. hsPAP gamma exhibits fundamental properties of a bona fide poly(A) polymerase, specificity for ATP, and cleavage and polyadenylation specificity factor/hexanucleotide-dependent polyadenylation activity. The catalytic parameters indicate similar catalytic efficiency to that of hsPAPII. Mutational analysis and sequence comparison revealed that hsPAP gamma and hsPAPII have similar organization of structural and functional domains. hsPAP gamma contains a U1A protein-interacting region in its C terminus, and PAP gamma activity can be inhibited, as can hsPAPII, by the U1A protein. hsPAPgamma is restricted to the nucleus as revealed by in situ staining and by transfection experiments. Based on these studies, it is obvious that multiple isoforms of PAP are generated by three distinct mechanisms: gene duplication, alternative RNA processing, and post-translational modification. The exclusive nuclear localization of hsPAP gamma establishes that multiple forms of PAP are unevenly distributed in the cell, implying specialized roles for the various isoforms (Kyriakopoulou, 2001).

Poly(A) polymerase activity is regulated by protein interactions with PAP regulatory domain proteins

The 3' ends of nearly all eukaryotic pre-mRNAs undergo cleavage and polyadenylation, thereby acquiring a poly(A) tail added by the enzyme poly(A) polymerase (PAP). Two well-characterized examples of regulated poly(A) tail addition in the nucleus consist of spliceosomal proteins, either the U1A or U170K proteins, binding to the pre-mRNA and inhibiting PAP via their PAP regulatory domains (PRDs). These two proteins are the only known examples of this type of gene regulation. On the basis of sequence comparisons, it was predicted that many other proteins, including some members of the SR family of splicing proteins, contain functional PRDs. The putative PRDs found in the SR domains of the SR proteins SRP75 and U2AF65, via fusion to a heterologous MS2 RNA binding protein, specifically and efficiently inhibit PAP in vitro and pre-mRNA polyadenylation in vitro and in vivo. A similar region from the SR domain of SRP40 does not exhibit these activities, indicating that this is not a general property of SR domains. The polyadenylation- and PAP-inhibitory activity of a given polypeptide can be accurately predicted based on sequence similarity to known PRDs and can be measured even if the polypeptides' RNA target is unknown. These results also indicate that PRDs function as part of a network of interactions within the pre-mRNA processing complex and suggest that this type of regulation will be more widespread than previously thought (Ko, 2002).

Selective stabilization of mammalian microRNAs by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2

The steady-state levels of microRNAs (miRNAs) and their activities are regulated by the post-transcriptional processes. It is known that 3' ends of several miRNAs undergo post-dicing adenylation or uridylation. The liver-specific miR-122 from human hepatocytes and mouse livers. Direct analysis by mass spectrometry revealed that one variant of miR-122 has a 3'-terminal adenosine that is introduced after processing by Dicer. GLD-2, which is a regulatory cytoplasmic poly(A) polymerase, was demonstrated to be responsible for the 3'-terminal adenylation of miR-122 after unwinding of the miR-122/miR-122* duplex. In livers from GLD-2-null mice, the steady-state level of the mature form of miR-122 was specifically lower than in heterozygous mice, whereas no reduction of pre-miR-122 was observed, demonstrating that 3'-terminal adenylation by GLD-2 is required for the selective stabilization of miR-122 in the liver (Katoh, 2009).

Hit and run versus long-term activation of PARP-1 by its different domains fine-tunes nuclear processes

Poly(ADP-ribose) polymerase 1 (PARP-1; see Drosophila Hiiragi) is a multidomain multifunctional nuclear enzyme involved in the regulation of the chromatin structure and transcription. PARP-1 consists of three functional domains: the N-terminal DNA-binding domain (DBD) containing three zinc fingers, the automodification domain (A), and the C-terminal domain, which includes the protein interacting WGR domain (W) and the catalytic (Cat) subdomain responsible for the poly(ADP ribosyl)ating reaction. The mechanisms coordinating the functions of these domains and determining the positioning of PARP-1 in chromatin remain unknown. Using multiple deletional isoforms of PARP-1, lacking one or another of its three domains, as well as consisting of only one of those domains, this study demonstrates that different functions of PARP-1 are coordinated by interactions among these domains and their targets. Interaction between the DBD and damaged DNA leads to a short-term binding and activation of PARP-1. This "hit and run" activation of PARP-1 initiates the DNA repair pathway at a specific point. The long-term chromatin loosening required to sustain transcription takes place when the C-terminal domain of PARP-1 binds to chromatin by interacting with histone H4 in the nucleosome. This long-term activation of PARP-1 results in a continuous accumulation of pADPr, which maintains chromatin in the loosened state around a certain locus so that the transcription machinery has continuous access to DNA. Cooperation between the DBD and C-terminal domain occurs in response to heat shock (HS), allowing PARP-1 to scan chromatin for specific binding sites (Thomas, 2019).

REFERENCES

Search PubMed for articles about Drosophila hiiragi

Ballantyne, S., Bilger, A., Astrom, J., Virtanen, A. and Wickens, M. (1995). Poly(A) polymerases in the nucleus and cytoplasm of frog oocytes: dynamic changes during oocyte maturation and early development. RNA 1: 64-78. 7489490

Benoit, B., et al. (1999). The Drosophila poly(A)-binding protein II is ubiquitous throughout Drosophila development and has the same function in mRNA polyadenylation as its bovine homolog in vitro. Nucleic Acids Res. 27: 3771-3778. 10481015

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 Citation: 18434412

Bienroth, S., Keller, W. and Wahle, E. (1993). Assembly of a processive mRNA polyadenylation complex. EMBO J. 12: 585-594. 8440247

Bilger, A., Fox, C. A., Wahle, E. and Wickens, M. (1994) Nuclear polyadenylation factors recognize cytoplasmic polyadenylation elements. Genes Dev. 8: 1106-1116. 7926790

Chang, J. S., Tan, L. and Schedl, P. (1999). The Drosophila CPEB homolog, Orb, is required for Oskar protein expression in oocytes. Dev. Biol. 215: 91-106. PubMed Citation: 10525352

Christofori, G. and Keller, W. (1989). Poly(A) polymerase purified from HeLa cell nuclear extract is required for both cleavage and polyadenylation of pre-mRNA in vitro. Mol. Cell. Biol. 9: 193-203. 2538718

Colgan, D. F., Murthy, K. G., Zhao, W., Prives, C. and Manley, J. L. (1998). Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites. EMBO J. 17: 1053-1062. 9463383

Dickson, K. S., Bilger, A., Ballantyne, S. and Wickens, M. P. (1999). The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation. Mol. Cell. Biol. 19: 5707-5717. 10409759

Dickson, K. S., Thompson, S. R., Gray, N. K. and Wickens, M. (2001). Poly(A) polymerase and the regulation of cytoplasmic polyadenylation. J. Biol. Chem. 276: 41810-41816. 11551905

Gebauer, F. and Richter, J. D. (1995). Cloning and characterization of a Xenopus poly(A) polymerase. Mol. Cell. Biol. 15: 1422-1430. 7862135

Hake, L. E. and Richter, J. D. (1994). CPEB is a specificity factor that mediates cytoplasmic polyadenylation during Xenopus oocyte maturation. Cell 79: 617-627. 7954828

Juge, F., Zaessinger, S., Temme, C., Wahle, E., Simonelig, M. (2002). Control of poly(A) polymerase level is essential to cytoplasmic polyadenylation and early development in Drosophila. EMBO J. 21(23): 6603-13. 12456666

Kashiwabara, S., et al. (2000). Identification of a novel isoform of poly(A) polymerase, TPAP, specifically present in the cytoplasm of spermatogenic cells. Dev. Biol. 228: 106-115. 11087630

Katoh, T., et al. (2009). Selective stabilization of mammalian microRNAs by 3' adenylation mediated by the cytoplasmic poly(A) polymerase GLD-2. Genes Dev. 23(4): 433-8. PubMed Citation: 19240131

Ko, B., Gunderson, S. I. (2002). Identification of new poly(A) polymerase-inhibitory proteins capable of regulating pre-mRNA polyadenylation. J. Mol. Biol. 318(5): 1189-1206. 12083511

Kyriakopoulou, C. B., Nordvarg, H. and Virtanen, A. (2001). A novel nuclear human poly(A) polymerase, PAP. J. Biol. Chem., 276: 33504-33511. 11431479

Lee, Y. J., Lee, Y. and Chung, J. H. (2000). An intronless gene encoding a poly(A) polymerase is specifically expressed in testis. FEBS Lett. 487: 287-292. 11150526

Martin, G. and Keller, W. (1996). Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and catalytic domain, homologous to the family X polymerases and to other nucleotidyltransferases. EMBO J. 15: 2593-2603. 8665867

Mendez, R., et al. (2000). Phosphorylation of CPEB by Eg2 mediates the recruitment of CPSF into an active cytoplasmic polyadenylation complex. Mol. Cell, 6: 1253-1259. 11106762

Mendez, R. and Richter, J. D. (2001). Translational control by CPEB: a means to the end. Nat. Rev. Mol. Cell Biol. 2: 521-529. 11433366

Mount, S. M. and Salz, H. K. (2000). Pre-messenger RNA processing factors in the Drosophila genome. J. Cell Biol. 150 F37-F44. 10908584

Murata, T., Ogura, K., Murakami, R., Okano, H., and Yokoyama, K. K. (1996). hiiragi, a gene essential for wing development in Drosophila melanogaster, affects the Notch cascade. Genes Genet. Sys. 71: 251-258. 8979376

Murata, T., Nagaso, H., Kashiwabara, S., Baba, T., Okano, H. and Yokoyama, K. K. (2001). The hiiragi gene encodes a poly(A) polymerase, which controls the formation of the wing margin in Drosophila melanogaster. Dev. Biol. 233: 137-147. 11319863

Murthy, K. G. K. and Manley, J. L. (1995). The 160-kD subunit of human cleavage-polyadenylation specificity factor coordinates pre-mRNA 3'-end formation. Genes Dev. 9: 2672-2683. 7590244

Perumal, K., (2001). Purification, characterization and cloning of the cDNA of human signal recognition particle RNA 3'-adenylating enzyme. J. Biol. Chem. 276: 21791-21796. 11287430

Raabe, T., Bollum, F. J. and Manley, J. L. (1991). Primary structure and expression of bovine poly(A) polymerase. Nature 353: 229-234. 1896071

Salles, F. J., (1994). Coordinate initiation of Drosophila development by regulated polyadenylation of maternal messenger RNAs. Science 266: 1996-1999. 7801127

Schisa, J. A. and Strickland, S. (1998). Cytoplasmic polyadenylation of Toll mRNA is required for dorsal-ventral patterning in Drosophila embryogenesis. Development 125: 2995-3003. 9655821

Schmid, M., Küchler B. and Eckmann, C. R. (2009). Two conserved regulatory cytoplasmic poly(A) polymerases, GLD-4 and GLD-2, regulate meiotic progression in C. elegans. Genes Dev. 23(7): 824-36. PubMed Citation: 19339688

Schul, W., Driel, R. V. and Jong, L. D. (1998). A subset of poly(A) polymerase is concentrated at sites of RNA synthesis and is associated with domains enriched in splicing factors and poly(A) RNA. Exp. Cell Res. 238: 1-12. 9457051

Takagaki, Y., Ryner, L. C. and Manley, J. L. (1989). Four factors are required for 3'-end cleavage of pre-mRNAs. Genes Dev. 3: 1711-1724. 2558045

Thomas, C., Ji, Y., Wu, C., Datz, H., Boyle, C., MacLeod, B., Patel, S., Ampofo, M., Currie, M., Harbin, J., Pechenkina, K., Lodhi, N., Johnson, S. J. and Tulin, A. V. (2019). Hit and run versus long-term activation of PARP-1 by its different domains fine-tunes nuclear processes. Proc Natl Acad Sci U S A 116(20): 9941-9946. PubMed ID: 31028139

Thuresson, A. C., Astrom, J., Astrom, A., Gronvik, K. O. and Virtanen, A. (1994). Multiple forms of poly(A) polymerases in human cells. Proc. Natl Acad. Sci. 91: 979-983. 8302877

Topalian, S. L., (2001). Identification and functional characterization of neo-poly(A) polymerase, an RNA processing enzyme overexpressed in human tumors. Mol. Cell. Biol. 21: 5614-5623. 11463842

Wahle, E., Martin, G., Schiltz, E. and Keller, W. (1991a). Isolation and expression of cDNA clones encoding mammalian poly(A) polymerase. EMBO J. 10: 4251-4257. 1756732

Wahle, E. (1991b). A novel poly(A)-binding protein acts as a specificity factor in the second phase of messenger RNA polyadenylation. Cell 66: 759-768. 1878970

Wahle, E. (1995). Poly(A) tail length control is caused by termination of processive synthesis. J. Biol. Chem. 270: 2800-2808. 7852352

Wang, L., Eckmann, C. R., Kadyk, L. C., Wickens, M. and Kimble, J. (2002). A regulatory cytoplasmic poly(A) polymerase in Caenorhabditis elegans. Nature 419: 312-316. 12239571

Wreden, C., et al. (1997). Nanos and Pumilio establish embryonic polarity in Drosophila by promoting posterior deadenylation of hunchback mRNA. Development 124: 3015-3023. 9247343

Zhao, J., Hyman, L. and Moore, C. (1999). Formation of mRNA 3'-ends in eukaryotes: mechanism, regulation and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63: 405-445. 10357856

Zhao, W. and Manley, J. L. (1996). Complex alternative RNA processing generates an unexpected diversity of poly(A) polymerase isoforms. Mol. Cell. Biol. 16: 2378-2386. 8628305

date revised: 5 August 2011

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.