Nucleic Acids Research

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Nucleic Acids Research 27:3771-3778 (1999)
© 1999 Oxford University Press

Article

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

Béatrice Benoit, Annette Nemeth¹, Nathalie Aulner², Uwe Kühn¹, Martine Simonelig^a, Elmar Wahle¹ and Henri-Marc Bourbon²

Dynamique du Génome et Evolution, Institut Jacques Monod, 2 Place Jussieu, 75005 Paris, France, ¹Institut für Biochemie, Justus Liebig Universität Giessen, Heinrich Buff Ring 58, 35392 Giessen, Germany and ²Centre de Biologie du Développement, UMR 5544 du CNRS, Université Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The poly(A)-binding protein II (PABP2) is one of the polyadenylation factors required for proper 3"-end formation of mammalian mRNAs. We have cloned Pabp2, the gene encoding the Drosophila homolog of mammalian PABP2, 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, we show 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The 3"-end formation of virtually all eukaryotic mRNA precursors (pre-mRNAs) occurs in a two-step reaction, which includes endonucleolytic cleavage of pre-mRNA, followed by the addition of a poly(A) tail of 200–250 nt to the upstream cleavage product (reviewed in 1–4). In mammals, both poly(A) site selection and polyadenylation require a multicomponent complex whose assembly is dependent on the highly conserved AAUAAA polyadenylation signal found 10–35 nt upstream of the cleavage site. Most of the 3"-end processing factors have been purified from mammalian cells and cDNAs coding for them have been cloned (reviewed in 2). Accurate cleavage of mammalian pre-mRNA substrates in vitro requires five factors: cleavage and polyadenylation specificity factor (CPSF), which consists of four subunits and binds the AAUAAA element; cleavage stimulation factor (CstF), which consists of three subunits; cleavage factors I and II (CFI and CFII); poly(A) polymerase (PAP). Poly(A) addition to a ‘pre-cleaved’ RNA can be reconstituted from three purified factors: PAP, CPSF and poly(A)-binding protein II (PABP2). PAP by itself has a low affinity for RNA and is unable to recognize a pre-mRNA specifically. Specificity requires the AAUAAA element and CPSF (5,6), but even in the presence of CPSF, the activity of PAP remains weak. PAP activity is again stimulated by binding of PABP2 to the poly(A) tail when the tail has reached a length of 10 adenylate residues (7,8). 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 (9). PABP2 also appears to be involved in control of poly(A) tail length. When the poly(A) tail has reached a length of ~250 residues, its elongation is no longer processive and becomes slow and distributive. PABP2 binds the poly(A) tail stoichiometrically; therefore, it is thought to be involved in a counting mechanism that determines the number of residues in the poly(A) tail and allows processive elongation to terminate when the length of the tail is ~250 nt (10).

cDNA and genomic clones encoding bovine PABP2 were previously isolated (11). The sequence of cDNAs predicts a 306 amino acid protein with a calculated molecular weight of 33 kDa. This protein contains a single RNP-type consensus RNA-binding domain (12,13) between an acidic N-terminal and a basic C-terminal domain. Recently, cDNAs and genomic clones encoding PABP2 have also been isolated from mouse (14) and human (15). The protein is highly conserved between the three species. In contrast, the RNA-binding domain of PABP2 is clearly different from any of the four RNA-binding domains found in the cytoplasmic poly(A)-binding protein (PABP) (16,17).

To confirm the role of PABP2 as a key polyadenylation factor in vivo, we have started a functional analysis of the Drosophila melanogaster PABP2 protein. The Drosophila gene encoding PABP2 (Pabp2) produces several mRNAs which all encode the same protein. This protein is mostly nuclear and present ubiquitously throughout Drosophila development. In addition, we show that Drosophila PABP2 has the same function as its bovine homolog in reconstituted mammalian polyadenylation assays. This study is a first step toward a genetic analysis which would allow determination of the biological function of Drosophila PABP2.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cloning of Pabp2 genomic DNA and cDNAs
A D.melanogaster genomic library in EMBL3 (18) was screened with a probe corresponding to the 2C3 cDNA insert (19). One phage, 2G2, was recovered and subcloned into pBluescript II (Stratagene). A 3.7 kb NsiI–PstI subclone was sequenced on both strands by the chain termination method using Sequenase 2.0 (US Biochemical) and either T3, T7 or specific primers. The Pabp2 genomic sequence was deposited in GenBank (accession no. AF116341). Imaginal disc cDNAs were isolated by screening an oligo(dT)-primed imaginal disc cDNA library in ZAPII, provided by Konrad Basler (University of Zürich), with a probe corresponding to 144 bp of Pabp2 cDNA spanning the RNA-binding domain. Sequence data were analyzed with the FASTA, BLAST, BESTFIT and PRETTY programs of the GCG package.

RNA blots and RNase protection assays
RNA blots were performed as reported previously (20) using the wild-type stock Canton S. The RNase protection assays were performed as reported by Audibert and Simonelig (21), with total RNA from the Canton S stock. To produce the antisense probe a EagI–BbsI genomic fragment of Pabp2 (nt 1452–3068) was cloned into pBluescript II; the resulting clone was digested with BfaI (position 2040) and transcribed with T3 RNA polymerase. The probe was 1095 nt long and after hybridization at 42°C and RNase digestion, protected fragments with sizes of 597 nt for 2C3 and DD68 cDNAs, 424 nt for 2C7 cDNA, 393 nt for 2C1 cDNA and 273 nt for 2C2 and DD16 cDNAs were expected.

Expression and purification of Drosophila and bovine PABP2-His₆ proteins
The complete ORF of Drosophila Pabp2 was PCR amplified with an upstream primer introducing an NdeI site as part of the ATG initiation codon and a downstream primer introducing a BamHI site after the stop codon. The PCR product was digested with NdeI and BamHI and cloned into the T7 expression vector pGM10 (22). The ORF of bovine PABP2 was cut out as a NdeI–BamHI fragment from the expression construct described earlier (11) and cloned into pGM10. Both fusion proteins carry the sequence Met-Ala-His₆ at their N-termini. Transformed cells were grown as described (11), except ampicillin was substituted by 100 µg/ml carbenicillin and the culture medium was changed every 2 h to guarantee stringent selection conditions. The cells were harvested and resuspended in 50 mM Tris–HCl, pH 8.0, 300 mM KCl and stored at –70°C. While the cells were allowed to thaw, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin and 2 µg/ml pepstatin were added. The suspension was sonicated, centrifuged, and the supernatants were incubated overnight with 500 µl/l culture of a 50% slurry of Ni²⁺–NTA agarose (Qiagen) on a rotating platform in a cold room. The mix was packed into a column and washed with 10 column vol 50 mM Tris–HCl pH 8.0, 50 mM KCl, 10% (v/v) glycerol, 10 mM imidazole-HCl. The protein was eluted in 3 ml of the same buffer containing 250 mM imidazole-HCl. Drosophila PABP2-His₆ was further purified on a 1 ml MonoQ FPLC column and bovine PABP2-His₆ on a 1 ml MonoS FPLC column (Pharmacia).

Poly(A) binding and polyadenylation assays
Poly(A) binding activity was determined by a nitrocellulose filter-binding assay. A reaction mix with 5"-end-labeled RNA in filter-binding buffer (50 mM Tris–HCl pH 8.0, 100 mM KCl, 1 mM EDTA, 0.5 mM DTT) was assembled, divided into aliquots of 40 µl each, and the binding reaction was started by the addition of protein in a volume of 10 µl. For the K_d determination 0.7 nM A₁₄ was used and for the competition experiments 150 nM A₇₀ (concentrations refer to AMP). The incubation time was 30 min at room temperature. Immediately before use, each nitrocellulose filter (0.2 µm pore size; Schleicher & Schuell NC 20) was washed with 1 ml wash buffer (50 mM Tris–HCl pH 8.0, 100 mM KCl, 1 mM EDTA) supplemented with 4 µg/ml Escherichia coli rRNA, then 80% of the binding reaction was applied to the filter and the filter was washed with 5 ml ice-cold wash buffer. Bound radio­activity was counted in a liquid scintillation counter. Polyadenyl­ation assays were carried out as described (10), except the reaction volume was reduced to 15 µl. PABP2 and CPSF from calf thymus and recombinant bovine PAP were purified as described (10). RNA substrates were prepared as described (8,10).

Antibody preparation
For the generation of rabbit polyclonal antibodies, the recombinant Drosophila PABP2 protein was purified under denaturing conditions. Cells were resuspended in 8 M urea, 100 mM NaH₂PO₄, 10 mM Tris–HCl, pH 8.0, stirred for 2 h at room temperature, sonicated and centrifuged. An aliquot of 2 ml of a 50% slurry of Ni²⁺–NTA agarose was added to the supernatant for every liter of culture, and the mixture was incubated for 2 h on a rotating platform at room temperature and then packed into a column. The column was washed with 20 vol of the resuspension buffer and 60 vol of the same buffer adjusted to pH 6.3. The protein was eluted with this buffer containing 500 mM imidazole-HCl and dialyzed extensively against a series of buffers with decreasing concentrations of urea. The protein was concentrated in a Centricon 10 cartridge (Amicon), washed twice with 1 ml 50 mM NH₄HCO₃ and lyophilized. Antibodies were prepared by Eurogentec (Belgium). Antibody affinity purification was performed as follows. Approximately 500 µg purified, recombinant Drosophila PABP2 was dissolved in 1 ml of coupling buffer (0.2 M NaHCO₃, 0.5 M NaCl pH 8.3) and covalently linked to a 1 ml NHS-activated HiTrap column (Pharmacia). Activation, protein coupling and inactivation of remaining reactive groups were carried out according to the manufacturer’s protocols. After coupling, the column was equilibrated with PBS. Antibody-containing rabbit serum was centrifuged for 2 min at 14 000 g. An aliquot of 1.1 ml of the cleared serum was diluted with 4 ml PBS and passed over the column five times. After washing with 15 ml PBS, bound antibodies were eluted with 0.2 M glycine-HCl, pH 2.5. Fractions (1 ml) were collected into tubes containing 350 µl of ice-cold 1 M Tris–HCl and immediately mixed. After pooling of antibody-containing fractions, they were concentrated and the buffer was changed to PBS in a Centriplus 10 vial. Antibodies were stabilized by the addition of acetylated BSA to a final concentration of 0.1 mg/ml.

Western blots and antibody staining
The wild-type stock Canton S was used. For western blots, tissues were homogenized in 3x sample buffer (2% SDS, 0.125 M Tris–HCl pH 6.9, 5% ß-mercaptoethanol, 10% glycerol, bromophenol blue) and proteins were separated by SDS–PAGE. After electrotransfer to nitrocellulose, the blot was blocked in PBS, 0.5% Tween-20, 5% milk. The Drosophila PABP2 protein was detected using the affinity-purified antibody at a dilution of 1:1000 in PBS, 0.5% Tween-20, 5% milk and revealed using anti-rabbit Ig horseradish peroxidase (1:1500) and an ECL kit (Amersham). For immuno­staining, ovaries were fixed for 20 min in 4% para­formaldehyde, 0.5% NP-40 (200 µl) and heptane (800 µl), then washed for 3 min with methanol and three times with PBS. Tissues were blocked for 2 h in PBT (PBS, 1% BSA, 0.1% Triton X-100) and incubated overnight at 4°C with the affinity-purified antibody 1:250 in PBT. After four washes over 2 h in PBS, 0.1% BSA, 0.1% Triton X-100, 2% horse serum, tissues were incubated with preabsorbed FITC-conjugated anti-rabbit IgG (Institut Pasteur Production) 1:50 in PBS, 0.1% BSA, 0.1% Triton X-100 for 2 h at room temperature. Following four washes in PBS, 0.1% BSA, 0.1% Triton X-100 over 2 h and one wash in PBS, tissues were mounted in Citifluor. Embryos were dechorionated for 5 min in bleach and fixed for 30 min in 0.1 mM PIPES, 2 mM MgSO₄, 1 mM EGTA (2.1 ml), 37% formaldehyde (0.4 ml) and heptane (2.5 ml). Following devitellinization in heptane (3 ml) and methanol–EGTA (9:1) (3 ml), embryos were rehydrated in PBS, blocked for 2 h in PBT with 0.5 mg /ml RNase A at room temperature and treated like ovaries except that the affinity-purified antibody was at a dilution of 1:500. Embryos were then incubated for 3 min in PBS, 0.1% BSA, 0.1% Triton X-100 with 1 µg/ml propidium iodide, before being washed in PBS and mounted in Citifluor. Confocal microscopy was carried out using a MCR 600 Bio-Rad confocal microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Drosophila PABP2 protein
By a PCR-based approach, we previously cloned cDNAs encoding three novel putative RNA-binding proteins of the RNP-type superfamily from D.melanogaster (19). Database searches revealed that one of these proteins, referred to as Rox2, shares extensive similarity with the bovine polyadenyl­ation factor PABP2 (11). The two proteins show 58% identity and 74% similarity. On the basis of this homology, the rox2 gene was renamed Pabp2. An alignment of the PABP2 protein from Drosophila, cow, human and mouse is shown in Figure 1. The shorter size of the Drosophila protein (224 amino acids) compared to the mammalian homologs (306 amino acids in cow) results from the lack in Drosophila PABP2 of an N-terminal region rich in alanine, glycine and proline residues. This region contains, in particular, an alanine stretch (amino acids 2–11) which is missing in Drosophila PABP2. In addition to the RNP-type RNA-binding domain, which is well conserved (77% identity and 87% similarity between the bovine and Drosophila proteins), another portion of the protein (amino acids 41–72 in Drosophila) in the N-terminal region is strikingly conserved (81% identity and 97% similarity between the bovine and Drosophila proteins). This conserved region shows a regular seven-residue pattern in which hydrophobic residues are found at alternate intervals of three and four residues. This pattern is characteristic of -helical coiled-coil domains known to be involved in homotypic and/or heterotypic protein–protein interactions (23,24). Bovine PABP2 can form oligomers in vitro (11); therefore, the conservation of this N-terminal domain and its similarity to coiled-coil domains indicate that it could be involved in oligomerization of PABP2. Alternatively, it might mediate an interaction with a different protein, e.g. PAP. Finally, 10 out of 15 arginine residues present in the basic C-terminal domain of the bovine protein are conserved in its Drosophila homolog (Fig. 1). Most of these arginines are methylated in bovine PABP2 (25).

View larger version (72K): [in this window] [in a new window]   Figure 1. Comparison of Drosophila PABP2 to mammalian homologs. Alignment of the amino acid sequence of Drosophila PABP2 (dPABP2) with sequences of bovine (bPABP2), human (hPABP2) and mouse (mPABII) PABP2 (accession nos: Drosophila, L34934; bovine, X89969; human, AF026029; mouse, U93050). Plus signs indicate the positions of conserved residues shared by the four proteins. Colons represent similarities and single dots represent less similar amino acids. The domain similar to -helical coiled-coil domains and the RNP-type RNA-binding domain are boxed. Within the coiled-coil domain, hydrophobic residues present at conserved positions in the four 7-residue repeats are in bold. In the C-terminal domain, arginine residues which are conserved between mammals and Drosophila are in bold.

 
Organization and expression of the Drosophila Pabp2 gene
We used the largest cDNA isolated previously (2C3) (19) as a probe to screen a Drosophila genomic library in EMBL3. One phage, 2G2, which covers the whole Pabp2 transcribed sequence, was recovered (structure shown in Fig. 2A). A 3.7 kb NsiI–PstI fragment from 2G2, which spans the largest Pabp2 cDNA, was subcloned and sequenced entirely. ­Comparison of this sequence to that of Pabp2 cDNAs allowed determination of the structure of the gene (Fig. 2A). ­Drosophila genomic Southern blots showed that Pabp2 is in single copy in the Drosophila genome (data not shown). The four cDNAs isolated previously from an embryonic library (19) and two cDNAs newly isolated from an imaginal disc library are shown in Figure 2B. Three cDNAs (2C2, 2C7 and 2C3) contain an extra G residue at their 5"-end which is not present in the corres­ponding genomic sequence. This may reflect a cap structure (26), suggesting that these cDNAs could be complete at their 5"-end and that alternative transcription start sites, clustered within 38 bp, could be used (Fig. 2D).

View larger version (55K): [in this window] [in a new window]   Figure 2. Structural organization of Pabp2 genomic and cDNA clones. (A) Restriction map of the 2G2 clone and structure of the Pabp2 gene (E, EagI; H, HindIII; M, SmaI; N, NsiI; P, PstI; R, EcoRI; X, XhoI). Hatched boxes represent coding sequences and open boxes are 5"- and 3"-UTRs. (B) Schematic representation of Pabp2 cDNAs from imaginal discs (DD16 and DD68) and from 4–8 h embryos (2C2, 2C1, 2C7 and 2C3). The sizes of the clones and of their poly(A) tracts are indicated. (C) RNase protection assays with total RNA from ovaries, 4–16 h embryos and pupae. Positions of the protected fragments are indicated. Control was with 10 µg of tRNA. (D) Sequences of Pabp2 5"- and 3"-UTRs. Intron 5 is in lower case letters and the stop codon (TGA) is in bold. Arrows indicate the start and the end of each cDNA clone. Poly(A) tracts in the 3"-UTR are underlined and consensus poly(A) signals are boxed.

 
Figure 2A shows that the 5"- and 3"-untranslated regions (UTRs) are each interrupted by a single intron and that three introns occur in the coding sequence. Strikingly, the third intron of Pabp2 and the fifth intron of its bovine counterpart disrupt the corresponding protein sequences at the same ­position, namely after an AAG codon specifying Lys167 in Drosophila and Lys243 in cow (Fig. 1).

The six cDNAs that we have isolated differ in the length of their 3"-UTRs. All six have a poly(A) stretch at their 3"-end and a consensus polyadenylation signal upstream of this oligo(A) (Fig. 2D). This suggests that all cDNAs could have a mature 3"-end. However, like its bovine homolog, Pabp2 contains oligo(A) stretches in its 3"-UTR. cDNAs 2C2, DD16 and 2C7 stop upstream of one of these oligo(A) stretches (Fig. 2D) and could result from internal priming within these stretches. We performed RNase protection assays to determine whether 2C2/DD16 and 2C7 have a mature 3"-end (Fig. 2C). A probe comple­mentary to a region of the 3"-UTR (position 2040–3068) was hybridized to total RNA from ovaries, embryos and pupae. This probe spans three poly(A) sites mapped from the sequence of 2C2/DD16, 2C1 and 2C7 cDNAs. The size of the probe-protected fragments at different developmental stages confirms the utilization of these three poly(A) sites (Fig. 2C). Therefore, analysis of cDNAs and RNase protection assays indicate utilization of at least five poly(A) sites in Pabp2. mRNAs produced by utilization of these alternative poly(A) sites have different 3"-UTRs but encode the same protein.

Pabp2 mRNAs were examined at various developmental stages by northern blot using a fragment of cDNA 2C3 as a probe (see Fig. 2B). Consistent with the structure of Pabp2 cDNAs, several transcripts were detected (Fig. 3). Their size is in rough agreement with the size of the cDNAs. Two mRNA species (1.3 and 1.7 kb) are present at all stages. Two other mRNAs are also detected. The 1 kb species is abundant in ovaries, larvae and pupae and the 1.9 kb species is detected at all stages except in ovaries and early embryos. Production of these mRNAs could result from utilization of the alternative poly(A) sites and/or from retention of introns. In cow and human, the first and sixth introns of PABP2 appear to be retained in some cDNA clones (11,15). We tested, by RT–PCR using RNA from males, females or ovaries as template, whether introns could be retained in Pabp2 RNAs and we found that they are not (data not shown). This suggests that the different transcripts result from utilization of the alternative poly(A) sites and possibly from utilization of different transcription start sites.

View larger version (63K): [in this window] [in a new window]   Figure 3. mRNA profile of Pabp2 during Drosophila development. Northern blot of poly(A)+ RNA from ovaries, 0–2, 2–4 and 4–16 h embryos, third instar larvae, pupae, adult males and adult females. Hybridization was with an RNA probe spanning the 5"-half of the Pabp2 coding sequence (Fig. 2B). The blot was reprobed with the rp49 clone as a loading control.

 
Immunodetection of the PABP2 protein during Drosophila development
An antibody against the Drosophila PABP2 protein was raised in rabbit and affinity purified. The specificity of this antibody was tested by western blot using protein extracts from various developmental stages. Figure 4A shows that a single 33 kDa protein is revealed by the antibody at each stage. The cDNA sequence predicts a 25 kDa protein. A difference between the predicted and the measured molecular mass of bovine PABP2 was also found and is thought to result from the unusual amino acid composition of the protein (11).

View larger version (95K): [in this window] [in a new window]   Figure 4. Detection of PABP2 protein during Drosophila development. (A) Western blot with protein extracts from 0–2 and 0–16 h embryos, third instar larvae, pupae, adult males, adult females and ovaries. (B) Immunodetection of the PABP2 protein during oogenesis; germarium and stages 1 and 2, stages 4 and 5, stage 7, stages 8 and 9 and stage 10B egg chambers. (C–N) Double staining of embryos with anti-PABP2 antibody (green) and propidium iodide (red). Posterior of the embryos is to the right. (C) Freshly laid embryo; (D) preblastoderm; (E) syncytial blastoderm; (F) cellularized blastoderm. Zygotic transcription starts during the syncytial blastoderm stage. (G–I) Posterior pole of a syncytial blastoderm stage embryo which shows accumulation of PABP2 in nuclei. (G) Anti-PABP2 staining; (H) propidium iodide staining; (I) superimposition of both patterns. (J–L) Anterior pole of an embryo after cellularization which shows accumulation of PABP2 in nuclei and mitotic domain 1. (J) Anti-PABP2 staining; (K) propidium iodide staining; (L) superimposition of both patterns. (M–N) Higher magnification of mitotic domain 1 shown in (J–L). (M) Anti-PABP2 staining; (N) superimposition of anti-PABP2 and propidium iodide staining patterns. Condensation of chromosomes in prophase (arrows) and metaphase plates (arrowheads) are visible. (O) High magnification of cells in a cellularized embryo which shows the non-homogeneous staining of anti-PABP2 in nuclei.

 
Using this specific antibody, we examined the localization of the PABP2 protein during Drosophila development. Figure 4B shows the distribution of PABP2 during oogenesis. Adult ovaries contain both somatic and germline cells. In the germarium, each germline stem cell produces a progeny of 15 nurse cells and one oocyte. This cluster is surrounded by somatic follicle cells to form the egg chamber. Egg chambers at successive developmental stages are arrayed within an ovariole, later stages of development being located more posteriorly (27). The PABP2 protein is detected throughout oogenesis in germline and follicle cells, and it mostly accumulates in nuclei, including oocyte nuclei. PABP2 also appears to be mostly nuclear in the germarium, but starting with egg chamber formation, nuclear accumulation in nurse cells and oocyte is accompanied by a widespread distribution of the protein in the cytoplasm. At stage 10, the protein is abundant in nuclei of nurse cells, oocyte and follicle cells and in nurse cell cytoplasm, but its amount is lower in oocyte cytoplasm. This could indicate that protein that is transferred to the oocyte from the nurse cells is translocated to the nucleus.

The distribution of the PABP2 protein during embryogenesis (28) was examined by double staining with the anti-PABP2 antibody and propidium iodide to reveal DNA and is shown in Figure 4C–N. In freshly laid embryos, PABP2 is uniformly distributed in the cytoplasm (Fig. 4C). It then progressively concentrates in nuclei during the preblastoderm and syncytial blastoderm stages (Fig. 4D, E and G–I), before the onset of zygotic transcription. After cellularization, the protein is mostly nuclear (Fig. 4F and J–L). It remains mostly nuclear and appears to be present in all cells at later stages of embryogenesis and in larvae (data not shown). In Figure 4J–N the first mitotic domain after cellularization of the embryo is also visible (29). Figure 4M and N shows that, during mitosis, the amount of PABP2 appears to be reduced in prophase nuclei; during metaphase, PABP2 is found in the cytoplasm and a large amount of the protein remains close to the metaphase plate.

Immunodetection of PABP2 in HeLa cells indicates that the protein is present in the nucleus both in a widespread pattern and in more intensely stained speckles (30) which correspond to sites in the nucleus where splicing factors accumulate. Figure 4O shows that nuclear staining with anti-PABP2 in cellularized embryos is non-homogeneous and also appears as a widespread staining with more intense regions.

Biochemical characterization of Drosophila PABP2
Drosophila PABP2 was expressed in E.coli with an N-terminal His₆-tag. The protein was purified on a Ni²⁺–NTA column, followed by chromatography on a MonoQ column. The ­recombinant protein was active in RNA binding. In nitro­cellulose filter binding experiments, oligo(A) with a chain length of 14 nt was bound with an apparent K_d of 10 nM (data not shown). This is very similar to the affinity of His-tagged recombinant bovine PABP2. In experiments designed to examine the RNA binding specificity of Drosophila PABP2, poly(A) with a chain length of ~70 nt was used as a radiolabeled ligand and binding was competed with unlabeled polynucleo­tides (Fig. 5). Unlabeled poly(A) competed as expected, binding being reduced to 50% by the addition of an equimolar amount of competitor. Poly(G) competed as efficiently as poly(A). Escherichia coli rRNA was a weaker competitor by a factor of about 10, whereas poly(C) had no significant effect even at a 100-fold excess. Thus, Drosophila PABP2 binds specifically to purine polyribo­nucleo­tides, as was previously reported for ­mammalian PABP2 (7,8).

View larger version (18K): [in this window] [in a new window]   Figure 5. Specificity of poly(A) binding by Drosophila PABP2-His6. For the poly(A) binding experiment radioactively labeled A70 and unlabeled competitor RNA in the indicated amounts were mixed before the protein was added. Binding of Drosophila PABP2-His6 to A70 without competitor was set to 100%.

 
Drosophila PABP2 was also assayed for its ability to replace mammalian PABP2 in the in vitro polyadenylation system. Drosophila PABP2 stimulated recombinant bovine PAP when assayed in the absence of CPSF with a simple oligo(A) primer (data not shown). The Drosophila protein was also able to replace bovine PABP2 in a complete reconstituted system including both mammalian CPSF and recombinant mammalian PAP with a primer RNA containing an AAUAAA sequence as well as an oligo(A) tail. Under these conditions, authentic bovine PABP2, recombinant His-tagged bovine PABP2 and recombinant His-tagged Drosophila PABP2 all led to rapid and processive poly(A) tail extension, whereas the reaction lacking a PABP2 showed a slow elongation (Fig. 6, compare the 20 s time points). The Drosophila PABP2-containing reaction also exerted a length control similar to the mammalian protein: poly(A) tail extension was rapid up to a length of ~250 nt and then proceeded more slowly, as described in detail before (10) (Fig. 6).

View larger version (61K): [in this window] [in a new window]   Figure 6. Drosophila PABP2 stimulates bovine PAP. The assay was carried out with 4 fmol PAP, 60 fmol CPSF, 50 fmol L3pre(A)15 substrate and with or without 100 fmol PABP2 purified from calf thymus (bPABP2), 200 fmol bovine PABP2-His6 (bPABP2-His6) or 500 fmol Drosophila PABP2-His6 (dPABP2-His6), both expressed in E.coli. Reactions, assembled on ice in the absence of ATP, were prewarmed, started by the addition of ATP and stopped after the indicated times by the addition of SDS-containing buffer. After purification the reaction products were analyzed on a denaturing 10% polyacrylamide gel.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this paper, we show that the rox2 cDNA, which we cloned previously in a molecular screen to isolate new RNP-type RNA-binding proteins, encodes the Drosophila homolog of mammalian PABP2. Biochemical characterization of the Drosophila protein shows that it behaves as mammalian PABP2 in in vitro assays: it binds specifically to purine poly­ribonucleotides and is able to replace bovine PABP2 in a reconstituted mammalian polyadenylation system. In the presence of bovine CPSF, recombinant bovine PAP and a primer RNA containing an AAUAAA signal and an oligo(A) tail, Drosophila PABP2 leads to rapid and processive extension of the poly(A) tail up to 250 nt and then the reaction becomes slow. This indicates that Drosophila PABP2 is able to stimulate PAP and to control the poly(A) tail length in vitro, as does bovine PABP2. These results show that Drosophila PABP2 has the same functions as bovine PABP2 in vitro. Functionality of the heterologous combination of mammalian and Drosophila proteins is also of interest with respect to the identification of interaction surfaces. Sequence comparison of PABP2 from Drosophila and mammals indicates that the protein is highly conserved throughout its entire length apart from the N-terminus. Three domains can be identified in the protein. The first domain is a 32-residue sequence which shows similarity to coiled-coil domains and is the most conserved region in the protein (81% identity between cow and Drosophila). This region could be involved in oligomerization of PABP2 (11), as similar coiled-coil domains are known to be involved in homotypic or heterotypic protein–protein interactions (23). Alternatively, this domain might mediate an interaction with PAP. The RNP-type RNA-binding domain shows 77% identity between the bovine and Drosophila proteins. The third domain, at the C-terminus of the protein, is rich in arginine residues, most of which are conserved between Drosophila and mammals. This region contributes to RNA binding (A.Nemeth, U.Kühn and E.Wahle, unpublished data). The lack of conservation of the N-terminal part of the protein in Drosophila, as well as the shorter size of this region (40 residues versus 115 residues in cow) indicates that this part of the protein is not essential for function of PABP2 in vitro. In mammals, this region contains an alanine stretch the expansion of which is responsible for oculopharyngeal muscular dystrophy in humans (15). This part of the protein, which is lacking in Drosophila, could have a role in PABP2 function in vivo in mammals.

Cloning of the Pabp2 gene and analysis of its RNA pattern indicate that it produces at least four RNA species, the relative amounts of which vary at different developmental stages. Analysis of cDNAs reveals that these different RNA species do not result from alternative splicing but from utilization of alternative poly(A) sites. cDNAs we have isolated stop at five different positions within 480 nt in the 3"-UTR of Pabp2. These five poly(A) sites are preceded by poly(A) signals and their utilization has been confirmed by RNase protection assays (Fig. 2C and data not shown). Use of at least two alternative poly(A) sites has also been described for the mouse PABII gene (14). In mouse as in Drosophila, mRNAs produced by utilization of the different poly(A) sites encode the same protein, but vary in their 3"-UTRs; this could influence their stability or translatability. Four stretches of oligo(A) are present in the 3"-UTR of Pabp2; this is reminiscent of the bovine PABP2 3"-UTR, which contains six stretches of oligo(A). These oligo(A) stretches are potential binding sites for PABP2. Their conservation in Drosophila reinforces the hypothesis that an autoregulatory loop could regulate PABP2 expression (11). In contrast, retention of the first and last introns of the PABP2 message, which occurs in human (15) and in cow (11) and could also regulate the level of PABP2 protein, does not appear to be conserved in Drosophila.

Western blots and immunodetection of Drosophila PABP2 indicate that the protein is ubiquitously expressed in Drosophila and accumulates mostly in nuclei. Nuclear localization is in agreement with a role of PABP2 in poly­adenylation and the presence of the protein in all tissues suggests that it is involved in polyadenylation of most mRNAs. Immunodetection experiments did not allow us to exclude the possibility that a low amount of the protein is present in the cytoplasm of somatic cells. A low level of PABP2 was also detected in the cytoplasm of HeLa cells by electron micro­scopy (30). The presence of PABP2 in the cytoplasm would corroborate recent findings suggesting that PABP2 shuttles between nucleus and cytoplasm (31; A.Calado and M.Carmo-Fonseca, personal communication).

The distribution of PABP2 during embryogenesis suggests that the protein is provided maternally to the embryo since the protein is present before zygotic transcription. This is not unexpected as the protein should be present at the onset of zygotic transcription for 3"-end processing of zygotic mRNAs. These experiments also reveal that PABP2 is quite abundant in the cytoplasm of early embryos where cytoplasmic poly­adenylation takes place. Cytoplasmic elongation of maternal mRNA poly(A) tails allows the translational activation of these maternal mRNAs, which are stored as dormant untranslated mRNAs during oogenesis (32). In Drosophila, at least bicoid and Toll mRNAs have been shown to undergo this regulation which activates the production of Bicoid and Toll proteins during early embryogenesis, where they play a key role in the anterior and dorsal–ventral patterning systems, respectively (33,34). The presence of PABP2 in the cytoplasm of early embryos is consistent with a possible role of PABP2 in this regulatory process. To test this hypothesis and to analyze the role of PABP2 in poly(A) tail formation and in poly(A) tail length control in vivo, a genetic analysis of Drosophila Pabp2 is in progress.

	ACKNOWLEDGEMENTS

 
The work of A.N. and E.W. was initiated while they were at the Biozentrum, University of Basel. We are grateful to Diana Blank for expert technical assistance and Walter Keller for support. This work was supported by an ATIPE grant (no. 3) from the Centre National de la Recherche Scientifique and a grant from the Association pour la Recherche sur le Cancer (no. 1184 to H.M.B.), by the Deutsche Forschungsgemeinschaft (to E.W. and U.K.) and the Fonds der Chemischen Industrie (to E.W.) and by the Centre National de la Recherche Scientifique (UMR 9922), the Universities P. et M. Curie et D. Diderot and the Association pour la Recherche sur le Cancer (no. 9885 to M.S.). B.B. held an award from the Ministère de l’Enseignement Supérieur et de la Recherche.

	FOOTNOTES

 
^a To whom correspondence should addressed at present address. Tel: +33 4 99 61 99 59; Fax: +33 4 99 61 99 57; Email: Martine.Simonelig{at}igh.cnrs.frPresent addresses:Béatrice Benoit and Martine Simonelig, Génétique du Développement de la Drosophile, Institut de Génétique Humaine, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, FranceAnnette Nemeth, Uwe Kühn and Elmar Wahle, Institut für Biochemie, Universität Halle, 06099 Halle, Germany The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

DDBJ/EMBL/GenBank accession no. AF116341.

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Received August 6, 1999. Revised and Accepted August 13, 1999.

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