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First published online 23 April 2008
doi: 10.1242/dev.021444

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

¹ mRNA Regulation and Development, Institut de Génétique Humaine, CNRS UPR 1142, 141 rue de la Cardonille, 34396 Montpellier Cedex 5, France.
² Department of Biochemistry, 433 Babcock Drive, University of Wisconsin, Madison, WI 53706 1544, USA.

View larger version (39K): [in this window] [in a new window]   Fig. 1. GLD-2 genes and proteins in Drosophila. (A) Schematic of GLD-2 proteins from different species. Accession numbers are NP_572766 for Wisp, NP_651012 for CG5732-encoded protein, NP_491842 for C. elegans, AAT98005 for Xenopus and NP_776158 for human. The regions showing homology are in gray and black; percentage similarity with Wisp in the central (including catalytic) domain and in the PAP/25A domain are indicated. The mutation D1031A in the catalytic domain that precludes poly(A) polymerase activity, and the point mutation in wisp12-3147, are shown. (B) Schematic of wisp (CG15737) locus and mutant. Black boxes are exons. The arrow indicates the transcription start site. The three cDNAs LD18468, RE03648 and RE14825 were sequenced. RE03648 and RE14825 are full-length and start and end at identical nucleotides, whereas LD18468 is incomplete at both ends. The P-element (not drawn to scale) in wispKG5287 is shown. The insertion site was verified by sequencing. (C) Western blots with anti-Wisp, showing Wisp expression in females and ovaries and the lack of protein in wispKG5287, wisp40 and wisp89 mutant ovaries. Protein extracts were from 0.4 (lane 1) or 0.2 (lanes 2, 9) wild-type ovaries, from one (lanes 3-6) or 0.2 (lanes 7, 8) mutant ovaries, from 0.3 male (lane 10) or 0.1 female (lane 11), and from 20 wild-type embryos (right panel). -tubulin was used as a loading control. (D) Immunostaining of ovaries with anti-Wisp, showing cytoplasmic expression (green) throughout oogenesis and the lack of expression in wispKG5287 mutant ovaries. Nuclei are visualized with DAPI (blue). Anterior is oriented towards the left.

View larger version (53K): [in this window] [in a new window]   Fig. 2. wisp function in the Drosophila germline. (A-E) Meiotic arrest as visualized in embryos. Immunostaining of 0- to 20-minute wild-type embryos and 0- to 2-hour wispKG5287/Df(1)RA47 embryos with anti--tubulin (green) and DAPI (blue). (A) Wild-type embryo showing mitoses. (B) wispKG5287/Df(1)RA47 embryo showing one metaphase I anastral female spindle and one mitotic-like spindle with a centrosome, associated with the male pronucleus (arrow). (C,D) Examples of meiosis I spindle in wispKG5287/Df(1)RA47 embryos showing scattered chromosomes along the spindle (C), and asymmetric pools of chromosomes (D). Bottom panels are stained with DAPI alone. (E) Scoring of meiotic arrest as visualized with anti--tubulin and DAPI staining. (F-J) Meiotic defects in stage 14 oocytes visualized with anti--tubulin (green) and DAPI (blue). (F) Prometaphase I in wild-type stage 14 oocyte. (G) Wild-type-like metaphase I in wispKG5287/Df(1)RA47 embryo; the fourth chromosomes are separated from the main chromosome pool (metaphase I arrest in J). (H,I) Abnormal metaphase I spindles in wispKG5287/Df(1)RA47 oocytes. Bottom panels are stained with DAPI alone. (J) Scoring of meiotic figures as visualized with anti--tubulin and DAPI. (K,L) DAPI staining of stage 8 oocytes, showing that the karyosome is not affected in the wisp mutant. (K) Wild type; (L) wispKG5287/Df(1)RA47.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Poly(A) polymerase activity of Wisp in a tethering assay and during oogenesis. (A,B) Tethering assay in Xenopus oocytes. (A) Wisp and a control protein, HsGLD-2, were tethered to luciferase reporter mRNA using MS2. β-galactosidase-encoding mRNA lacking MS2 binding sites was used as an internal control. Translational stimulation was assayed by measuring luciferase activity. Wild-type and point-mutant (DA) proteins were expressed at similar levels, as determined by western blotting with anti-HA11 (bottom). (B) 130 nt, 32P-labeled RNA was injected and then purified from oocytes. (Lanes 1-4) Tethered HsGLD-2 and Wisp added long poly(A) tails onto labeled RNA. Active site mutations disrupted elongation. (Lanes 5-8) Tails added by wild-type Wisp were removed by RNase H/oligo(dT) treatment, confirming that they were poly(A). -, RNase H only; +, RNase H plus oligo(dT). (Lanes 9-12) RNAs elongated by Wisp were bound to an oligo(dT) column. -, RNAs that did not bind; +, RNAs that bound. (C) PAT assays measuring osk, CycB, nos and bcd poly(A) tail lengths in Drosophila egg chambers of different stages (germarium to stage 8, stages 9 and 10, and stage 14) from wild-type and wispKG5287 ovaries. sop mRNA, which encodes a ribosomal protein and is not regulated by cytoplasmic polyadenylation, was used as a control.

View larger version (58K): [in this window] [in a new window]   Fig. 4. cort mRNA is a meiotic target of Wisp. (A) PAT assays measuring cort poly(A) tail lengths in Drosophila egg chambers of progressive stages from wild-type and wispKG5287 ovaries. sop mRNA was used as a control. (B) Western blots showing that Cort protein levels are reduced in wispKG5287 stage 14 oocytes as compared with wild type. Protein extracts were from 30 egg chambers at stage 9-10 and from 15 oocytes at stage 14. (C) Western blots showing that CycA levels are higher in the ovaries and stage 14 oocytes of wispKG5287 than of wild-type. Protein extracts were from 0.5 ovary and 20 oocytes at stage 14. -tubulin was used as a loading control in B and C.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Cytoplasmic polyadenylation complexes in Drosophila ovaries. (A-D) Immunoprecipitations (IPs) were performed in ovary extracts either in the presence of RNase inhibitor or in the presence of RNase A (RNase). Co-immunoprecipitated proteins were identified by western blot. Mock IPs were with preimmune serum for Wisp and PAP IPs, with rabbit serum for BicC IP and with an irrelevant monoclonal antibody for Orb IP. Extract (1/20) prior to IP was loaded. (E) In vitro interaction assays showing that Orb directly interacts with recombinant GST-Wisp(702-1373), but not with GST-Wisp(11-547) or GST alone. Orb was revealed by western blot. 1/10 of in vitro synthesized Orb before the interaction assay was loaded (input). Protein extract from 0.5 ovary was also loaded (upper panel). Recombinant and GST proteins are shown (input, bottom panel).

View larger version (38K): [in this window] [in a new window]   Fig. 6. PAP has an earlier role in Drosophila oogenesis than Wisp. (A) Genetic interactions between orb and wisp and between orb and hrg. Females of the indicated genotype were crossed with wild-type males and the embryonic lethality of their progeny was scored. The percentage of embryonic lethality from orbmel mothers is increased in the presence of both heterozygous wisp or hrg mutants. hrg mutations only are able to dominantly increase the ventralization phenotype of orbmel. Note that collapsed embryos with normal dorsal appendages is a common phenotype of wisp mutant embryos. This phenotype suggests a defect in vitelline membrane cross-linking, an early event at egg activation. (B) Ovaries of orbmel single mutant females, or in the presence of wisp-/+ or hrg-/+ mutants, visualized with DAPI. Oogenesis progresses normally in orbmel, wisp89/+; orbmel and wisp40/+; orbmel females, but is blocked at stage 8 in hrgPAP21/+; orbmel ovaries. Fifty ovarioles of the indicated genotype were scored and the percentage of abnormal egg chambers arrested at stage 8 is indicated. For stage 8 arrests that were scored in orbmel or wisp-/+; orbmel ovaries, the oocyte was present. (C) Characterization of stage 8 arrest in hrgPAP21/+; orbmel ovaries. Immunostaining of ovaries with anti-C(3)G antibody (green) and DAPI (blue), showing the presence of the oocyte in orbmel and its absence in arrested hrgPAP21/+; orbmel egg chambers (arrow). (D) Osk protein accumulation is not affected in wisp mutant oocytes during mid-oogenesis. Immunostaining of wild-type and wispKG5287/Df(1)RA47 mutant ovaries with anti-Osk antibody, showing that Osk accumulation at the posterior pole is similar in wild-type and wisp mutant stage 10 oocytes.

View larger version (62K): [in this window] [in a new window]   Fig. 7. Role of Wisp in cytoplasmic polyadenylation in early Drosophila embryos. (A) PAT assays and RT-PCR of bcd, osk and nos mRNAs in embryos from 0-1, 1-2 and 2-3 hours of development, from wild-type, wispKG5287 or wispKG5287/Df(1)RA47 females. In the absence of Wisp, osk and nos mRNAs are destabilized and the poly(A) tails of bcd mRNA are not elongated in embryos. sop mRNA was used as a control. The sop RT-PCR is the loading control for bcd, osk and nos RT-PCR. For bcd mRNA, PAT assays from ovaries were loaded to show the poly(A) tail elongation between ovaries and early embryos in the wild type. (B) In situ hybridizations of 0- to 1-hour wild-type and wispKG5287/Df(1)RA47 embryos showing bcd, osk and nos mRNA. (C) Immunostaining of 0- to 1-hour embryos with anti-Bcd, anti-Nos and anti-Osk antibodies, showing that the lack of poly(A) tail elongation in wispKG5287/Df(1)RA47 mutant embryos, leading or not to mRNA decay, results in the lack of corresponding proteins. Note that the lack of nos mRNA/protein at the posterior pole could also result from the lack of Osk protein, as Osk is required for nos mRNA stabilization.

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