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Ypsilon Schachtel, a Drosophila Y-box protein, acts antagonistically to Orb in the oskar mRNA localization and translation pathway

¹ Columbia University, Department of Biological Sciences, 602 Fairchild Building, 1212 Amsterdam Avenue, New York, NY 10027, USA
² Carnegie Institute of Washington, Department of Embryology, 115 W University Parkway, Baltimore, MD 21210, USA

View larger version (17K): [in a new window]   Fig. 1. A physical map of the yps gene, and the ypsJM2 deletion mutation. yps was mapped to polytene region 68F3-F7 by chromosome in situ hybridization. Three deletions that uncover yps are indicated: Df(3L)vin4, Df(3L)vin7 and Df(3L)BK9. Alignment of known yps cDNAs with genomic sequence predicts the exon structure of yps shown in the diagram. Each division of the black line represents 100 bp of genomic DNA, while the thick green and blue boxes represent exons. The precise locations of the 5' end of the transcripts have not been mapped. The cold shock domain (CSD) is shown in light blue, and the start (ATG) and stop (TAG) codons are marked. The EP(3)3078 insertion (represented by a red triangle) was mapped 259 bp upstream of the start codon (BDGP). The limits of the ypsJM2 deletion are represented by the blue line at the bottom. The positions of PCR primers used for the excision screen (see text) are indicated by the red arrows labeled a-d. The chromosome drawing is reproduced after the drawings of Bridges (Bridges, 1941).

View larger version (24K): [in a new window]   Fig. 2. yps expression in ovaries. (A) A northern blot of ovary poly(A+)RNA probed with a yps cDNA. Two forms of the yps transcript are detected in wild-type ovaries (lane WT): a minor band of approximately 2.3 kb and a major broad band of approximately 1.7 kb, which may actually represent several transcripts of similar size. The positions of RNA molecular mass markers (M) are indicated on the right. In ovary poly(A+) mRNA from ypsJM2/Df(3L)BK9 females, the yps mRNAs are truncated and present at greatly reduced levels (lane ypsJM2). rp49 serves as a loading control for both samples. (B) Whole-mount in situ hybridization of a yps cDNA to wild-type ovaries. yps is expressed in the germ cells from early oogenesis on, and its transcripts are distributed evenly throughout the nurse cells and can be detected at a low level in the oocyte. yps is also expressed at a low level in the follicle cells. (C) A western blot of ovary protein, probed with an antibody to the N terminus of Yps (a region that includes the CSD) reveals a single protein isoform migrating at approximately 50 kDa (lane WT). In protein extracted from ypsJM2/Df(3L)BK9 ovaries (lane ypsJM2), Yps is not detected.

View larger version (66K): [in a new window]   Fig. 3. Yps and Exu proteins are localized independently of one another. Ovaries in A and B are from female flies carrying a gfp-exu transgene. (A) GFP-Exu in control egg chambers illustrates the wild-type localization state of the protein. Exu accumulates in the oocyte from early oogenesis, and during mid-oogenesis becomes concentrated at the anterior and posterior poles of the oocyte. Within the nurse cells, Exu is localized to cytoplasmic particles. (B) GFP-Exu is expressed and localized normally in ypsJM2/Df(3L)BK9 ovaries. (C) Control ovaries labeled with an antibody to Yps reveal that, like Exu, Yps accumulates early in the oocyte, becomes concentrated at the anterior and posterior poles of the oocyte during mid-oogenesis, and is associated with particles in the nurse cell cytoplasm. Yps is also expressed in the follicle cells, unlike Exu. (D) In exuSC02/exuSC02 ovaries, Yps is expressed and localized normally.

View larger version (87K): [in a new window]   Fig. 4. ypsJM2 rescues the osk mRNA localization defects of orbmel/orbF303 females. In wild-type ovaries, osk mRNA accumulates in the oocytes of young egg chambers (stages 1-7), is transiently localized to the anterior of the oocyte in stage-8 to early stage-9 egg chambers, and then becomes localized to the posterior pole of the oocyte by late 9. (A) Egg chambers from control (w1118) ovaries. In the stage-10 egg chamber on the right, osk mRNA is localized at the posterior of the oocyte. (B) ypsJM2/ypsJM2egg chambers. osk mRNA accumulates normally in the oocytes of young-stage egg chambers on the left. In the stage-10 egg chamber in the center of the panel, osk mRNA is localized normally to the posterior pole of the oocyte. (C) orbmel/orbF303 egg chambers. In the stage-10 egg chamber in the center of the panel, osk mRNA is not localized at the posterior pole of the oocyte. (D) ypsJM2orbmel/ypsJM2orbF303egg chambers. In the stage-10 egg chamber on the right, osk mRNA is localized at the posterior pole of the oocyte, although its level is reduced compared to wild type.

View larger version (72K): [in a new window]   Fig. 5. ypsJM2 rescues orb-associated defects in osk protein expression, but does not alter the expression or localization of Orb protein. The ovaries in A-D were labeled with an antibody to the Osk protein. Arrows in A-D indicate the posterior poles of stage-10 oocytes. (A) w1118 (control) egg chambers. Osk protein is present at the posterior pole of the stage-10 oocyte. (B) An identical pattern is observed in ypsJM2/ypsJM2 egg chambers. (C) In orbmel/orbF303 mutant egg chambers, Osk protein is not detected at the posterior pole of the oocyte. (D) In many ypsJM2orbmel/ypsJM2orbF303 doubly mutant egg chambers, Osk protein is present, localized at the posterior pole. The ovaries shown in A-D were also labeled with an antibody to the Orb protein (E-H). (E) In control egg chambers, Orb concentrates in the oocyte from early oogenesis, and is present at lower levels in the nurse cells. During mid-oogenesis the protein accumulates at the anterior and posterior (arrow) poles of the oocyte. (F) An identical pattern is observed in ypsJM2/ypsJM2 egg chambers. (G) In orbmel/orbF303 egg chambers, Orb accumulates in the nurse cells to a higher level than in wild type. During mid-oogenesis, Orb is particularly concentrated in the nurse cells that border the oocyte (arrow). Orb rarely accumulates at the anterior or posterior poles of the oocyte. (H) In ypsJM2orbmel/ypsJM2orbF303 ovaries, the distribution of Orb is identical to orbmel/orbF303 ovaries.

View larger version (73K): [in a new window]   Fig. 6. ypsJM2 does not rescue orb-associated defects in grk mRNA localization. (A) In w1118 (control) mid-stage egg chambers, grk mRNA is localized to the dorsal anterior corner of the oocyte. (B) An identical pattern is observed in ypsJM2/ypsJM2 egg chambers. (C) In orbmel/orbF303 egg chambers, grk mRNA is spread along the entire anterior margin of the oocyte. (D) In ypsJM2orbmel/ypsJM2orbF303 doubly mutant egg chambers, grk mRNA localization is identical to orbmel/orbF303 egg chambers.

View larger version (42K): [in a new window]   Fig. 7. Orb coimmunoprecipitates with Exu and Yps in an RNAse-sensitive manner. (A) Immunoblot for the presence of Orb in Exu and Yps immunoprecipitates from GFP-Exu extracts using anti-GFP (GFP), anti-Yps (YPS) or rabbit IgG antibodies. Orb protein specifically coimmunoprecipitates with both Exu and Yps. (B) Immunoblot for the presence of Orb in Exu and Yps immunoprecipitates from RNAse-treated GFP-Exu extracts using anti-GFP (GFP), anti-Yps (YPS) or rabbit IgG antibodies. Orb protein does not coimmunoprecipitate with either Exu or Yps when RNAse-treated extracts are used, indicating that RNA is required for Orb to associate with Exu and Yps. In both panels, the right lane shows ovary extract prior to immunoprecipitation.