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First published online July 11, 2006
doi: 10.1242/10.1242/dev.02456

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A translation-independent role of oskar RNA in early Drosophila oogenesis

Andreas Jenny^1,*,, Olivier Hachet^1,,, Péter Závorszky^1,2, Anna Cyrklaff¹, Matthew D. J. Weston^3,, Daniel St Johnston³, Miklós Erdélyi^2,¶ and Anne Ephrussi^1,¶

¹ European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
² Biological Research Center of the Hungarian Academy of Sciences, Institute of Genetics, H-6701 Szeged, POB 521, Temesvari krt. 62, Hungary.
³ The Wellcome Trust/Cancer Research UK Gurdon Institute & the Department of Genetics, University of Cambridge, Tennis Court Rd, Cambridge CB2 1QN, UK.

View larger version (62K): [in a new window]   Fig. 1. Characterization of the oskA87 and osk187 alleles. (A) Schematic representation of the oskar locus and the insertion points of the transposable elements in the new oskar alleles. E1 through E4 represent the oskar exons. The first exon contains a 15 nucleotide untranslated region. In oskA87, a ZAM retrotransposable element is inserted 51 bp upstream of the first intron, in reverse orientation relative to oskar. In osk187, an I element is inserted 534 bp upstream of the oskar transcription initiation site, in the same orientation as oskar. (B,C) Northern blot and RT-PCR analysis of oskar mRNA in the new oskar mutants. (B) Northern blot: WT1 sample is RNA from wild-type (Oregon R) egg chambers of stages 1 to 14, and WT2 from wild-type egg chambers of stages 1 to 7-8. The eglRC12 sample represents ovaries whose development arrested during oogenesis at stages similar to the new oskar mutants. osk54 and osk84 are nonsense alleles of oskar (Kim-Ha et al., 1991). Only trace amounts of oskar mRNA are detected in homozygous osk187 egg chambers and no oskar mRNA is detected in osk187/Df(3R)pXT103 egg chambers. (C) RT-PCR: in contrast to bcd (upper panel), no oskar mRNA is detected by RT-PCR in oskA87/Df(3R)pXT103 egg chambers (lower panel, lane 2) while a 126 bp band is detected in a control amplification from nosA10/Df(3R)pXT103 (lower panel, lane 1), an allele of nanos originating from the same genetic background as oskA87. Lanes 3: negative controls without addition of template. (D) oskar RNA in situ hybridization in osk187 and oskA87. Fluorescently-labeled antisense oskar RNA probe was used to visualize the presence of endogenous oskar RNA in stage 3-4 wild-type, osk187/Df(3R)pXT103 and oskA87/Df(3R)pXT103 mutant egg chambers. Compared with wild type, considerably less or no oskar RNA is detected in osk187/Df(3R)pXT103 and in oskA87/Df(3R)pXT103 egg chambers, respectively.

View larger version (55K): [in a new window]   Fig. 2. An oocyte is specified in oskA87/Df(3R)pXT103. (A,B) Whole mount antibody staining of oskA87/Df(3R)pXT103 ovaries (B) shows that Staufen is not enriched in the oskA87mRNA null oocytes compared with wild type (A). (C-J) In contrast, the oocyte markers BicD (Suter and Steward, 1991), Orb (Lantz et al., 1994), Bruno (Webster et al., 1997), and Par-1 (Shulman et al., 2000; Tomancak et al., 2000) accumulate in a single, posterior cell in both wild-type (C,E,G,I) and oskA87/Df(3R)pXT103 (D,F,H,J) egg chambers, indicating that early steps in oocyte specification occur normally in the mutant. (K,L) DAPI staining of chromatin in stage 4 wild-type (K) and oskA87/Df(3R)pXT103 (L) egg chambers. The compact structure of the karyosome (arrow) is detected within the wild-type oocyte nucleus. oskA87/Df(3R)pXT103 mutant oocytes show a fragmented karyosome structure (arrow).

View larger version (25K): [in a new window]   Fig. 3. The oskar 3'UTR is sufficient to rescue the oogenesis arrest phenotype of oskar RNA null alleles. (A,C) Schematic of oskar alleles and transgenes. Solid black bars represent the oskar promoter and 5'UTR (left) and the 3'UTR (right). M1 and M2 are the two translation initiation sites of oskar. E1 through E4 indicate the oskar exons. Black dots in osk54, osk84, osk346 and P(mM1 mM2stop) transcripts show the positions of the stop codons in the nonsense alleles and the transgene. P(osk+) is a wild-type oskar transgene that encodes both the long and short Oskar isoforms and fully rescues the oskar strong loss of function alleles (Markussen et al., 1995). Black dots at M1 and M2 in P(oskM1L), P(oskM139L), P(mM1 mM2stop) and P(mM1SLmM2) indicate the mutated translation initiation sites. HP7 represents the hairpin loop sequence (Kozak, 1989a) inserted between M1 and M2, and the grey dot shows the frame-shift mutation inserted in P(mM1SLmM2). (C) Light grey bars represent the UAS promoter. UAS oskWT expresses the oskar gene (including introns) under the control of the yeast UAS promoter. UAS oski(1,2,3) expresses an oskar RNA whose three introns have been deleted (dark grey bar), under control of the UAS promoter. Note that all UAS-driven transcripts contain an intron derived from the pUASp vector. Dashed line in UAS osk-K10 indicates the K10 3'UTR. UAS osk3'UTR expresses only the 3'UTR of oskar without any oskar coding region under control of UAS. To ensure continued high-level expression throughout oogenesis, expression of all UAS transgenes was driven by pCOG:Gal4VP16 and nanos:Gal4VP16 simultaneously. Flies were grown at 25oC. In the case of the UAS osk-K10 transgene, rescue was also tested at 29oC, at which Gal4-induced expression is maximal. (B,D) Average number and standard deviation produced daily by females of the genotype indicated underneath (normalized to the average number of eggs laid by wild-type females). See text for details. (B) Grey bar: Oregon-R control. Black and white bars: alleles or transgenes in oskA87/Df(3R)pXT103 and osk187/Df(3R)pXT103 background, respectively. (D) Grey bar: w1118 control. Black bars: transgenes in oskA87/Df(3R)pXT103 background.

View larger version (46K): [in a new window]   Fig. 4. UAS osk-K10 produces Oskar protein before oogenesis arrest in oskA87/Df(3R)pXT103. (A) Scheme showing transgene structure and priming sites of PCR primers used for Real-time PCR. Large blue boxes represent oskar coding region. Grey and orange thin boxes represent oskar and K10 UTRs, respectively. Thin lines correspond to introns. Oskar specific primers are in red, K10 specific primer is in green. (B) Genotypes of ovaries analyzed. Numbers correspond to numbers in panels A, C. (C) Two percent agarose gel showing end products of Real-time PCR reactions performed in triplicate using cDNA of early stage ovaries of genotype numbered on top. bicoid (bcd) was used for normalization. UAS oskK10 is about 15 fold less abundant than UAS osk WT. NTC: non-template control. (D) Antibody staining of non-rescued ovaries from females expressing UAS oskK10 (genotype #1) show that Oskar protein (green) is expressed as early as in germaria. F-actin and DNA are in red and blue, respectively.

View larger version (94K): [in a new window]   Fig. 5. The oskar 3'UTR is sufficient for Staufen transport to the oocyte, but not for oskar RNA localization at the posterior pole. (A-C) Staufen revealed by immunofluorescence in stage 4 oskA87/Df(3R)pXT103 egg chambers expressing pCogGal4:VP16;NosGal4:VP16-driven UAS oskWT (A), UAS oskK10 (B), and UAS osk3'UTR (C). (D) The karyosome defect is rescued in egg chambers expressing pCogGal4:VP16;NosGal4:VP16-driven UAS osk3'UTR. White arrow marks an intact karyosome revealed by DAPI stain. (E-H) Top and bottom panels show stage 10 oskA87/Df(3R)pXT103 egg chambers expressing UAS-driven oskWT and osk3'UTR, respectively. (E,F) Staufen detected by immunofluorescence is shown in green; DAPI staining is in red. (G,H): oskar RNA detected by fluorescent in situ hybridization is shown in red; DAPI staining is in blue.