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).

GENE STRUCTURE

The Drosophila PAP-encoding gene was identified by screening a Drosophila genomic library with a bovine PAP cDNA (Wahle, 1991). One positive phage was isolated. Several subclones of this phage were used to screen cDNA libraries from 0- 3 h and 12- 24 h embryos, and ovaries. Restriction mapping and partial sequencing of 69 positive cDNAs as well as two expressed sequence tags (ESTs) from the Berkeley Drosophila Genome Project (LD11853 and LD05439) showed that the PAP-encoding gene produces three different mRNAs that arise from utilization of two alternative transcription start sites and two alternative poly(A) sites. No alternative splicing was found in the coding sequence and this gene was found to encode a single protein. In addition, no paralogous gene was found in the Drosophila genome either by Southern blot hybridization or by examination of the Drosophila genome sequence. Therefore, a single PAP, which is encoded by the hrg gene, is produced in Drosophila. Northern analysis shows that the two largest mRNAs (3.9 and 3.6 kb) are present at all development stages. The shortest mRNA (3.2 kb) arising from utilization of a downstream transcription start site is specific to early embryogenesis and strongly accumulates in 1.5- 3 h embryos. Visualization of hrg mRNAs in embryos by in situ hybridization correlates with a burst of transcription of an embryo-specific transcript before cellularized blastoderm (Juge, 2002).

cDNA clone length - 3835

Bases in 5' UTR - 708

Exons - 9

Bases in 3' UTR - 1174

PROTEIN STRUCTURE

Amino Acids - 659

Structural Domains

The deduced amino acid sequence of the Hrg protein includes a catalytic domain at the amino terminus [68.0% homology to amino acids (a.a.) 59 to 186 in bovine PAP] (Martin, 1996) and a region that was strongly homologous (69.7%) to bovine PAP in the central region of the protein (a.a. 187 to 422 in bovine PAP). The primer-binding region, which also includes a putative nuclear-localization signal sequence (a.a. 488 to 508), exhibits somewhat weaker homology to bovine PAP (47.6%) (Murata, 2001).

The predicted protein of 659 residues is 56% identical and 70% similar to bovine PAP. The overall identity between Drosophila PAP and each of the three human proteins (PAP II, neo-PAP and TPAP) is similar (45%- 50%). The N-terminal two-thirds of PAP that contain the catalytic core (67- 190) are well conserved between Drosophila and human. The C-terminal region is more divergent, except for a short well-conserved domain (465- 537) (Martin, 1996)that contains a primer-binding domain and a nuclear localization signal (Juge, 2002).

date revised: 17 January 2002

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