Cup is an eIF4E binding protein required for both the translational repression of oskar and the recruitment of Barentsz

Abstract

In Drosophila oocytes, precise localization of the posterior determinant, Oskar, is required for posterior patterning. This precision is accomplished by a localization-dependent translational control mechanism that ensures translation of only correctly localized oskar transcripts. Although progress has been made in identifying localization factors and translational repressors of oskar, none of the known components of the oskar complex is required for both processes. Here, we report the identification of Cup as a novel component of the oskar RNP complex. cup is required for oskar mRNA localization and is necessary to recruit the plus end-directed microtubule transport factor Barentsz to the complex. Surprisingly, Cup is also required to repress the translation of oskar. Furthermore, eukaryotic initiation factor 4E (eIF4E) is localized within the oocyte in a cup-dependent manner and binds directly to Cup in vitro. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. We propose that Cup coordinates localization with translation.

Figure 1.

Cup is a component of the oskar RNP complex. (A) Diagram showing the stage specific movements of oskar mRNA and the corresponding changes in Oskar translation and microtubule polarity. oskar mRNA, green; Oskar protein, blue; microtubules (MT), red. (B) Immunoblot for Cup (arrow) of immunoprecipitates from GFP-Exu extract using α-GFP (GFP), α-Yps (YPS), or rabbit IgG (IgG) antibodies. (C) Cup protein (green; arrows) is concentrated in at the posterior of the developing oocyte in stages 1–6 (stage 5 is shown; actin is in red). (D) Cup transiently accumulates at the anterior of the oocyte during stages 7 and 8 (stage 7 is shown). (E) Cup then accumulates at the posterior pole during stages 9 and 10 (stage 9 is shown). (F–H) Cup and Yps colocalize in cytoplasmic particles in nurse cells from stage 8 egg chambers. (F) α-Cup staining. (G) α-Yps staining. (H) Merged image. Cup is in red and Yps is in green. (I–K) Cup and Btz colocalize in cytoplasmic particles in nurse cells from stage 8 egg chambers. (I) α-Cup staining. (J) α-Btz staining. (H) Merged image. Cup is in red and Btz is in green. Bars, 10 μm.

Figure 2.

Cup mutations specifically disrupt oskar mRNA localization and Btz recruitment. Localization of oskar mRNA in ovaries from yw females during stage 4 (A), stage 7 (B), and stage 10 (C). Localization of oskar mRNA in cup ¹/cup⁴⁵⁰⁶ females during stage 5 (D), stage 8 (E), and stage 9 (F). oskar mRNA is distributed along the cortex of the oocyte in stages 8–10 in cup ¹/cup⁴⁵⁰⁶ egg chambers. (G) Btz (green) is localized to the nuclear envelope as well as the posterior pole of the oocyte (arrow) in yw egg chambers. (H) In cup ¹/cup⁴⁵⁰⁶ egg chambers, Btz accumulates at the nuclear envelope, but is only weakly present at the posterior pole of the oocyte (arrow). Yps (green) is localized normally to the posterior pole of the oocyte in both yw (I) and cup ¹/cup⁴⁵⁰⁶ (J) egg chambers. Actin is in red. Bars, 10 μm.

Figure 3.

Cup is required for translational repression of oskar mRNA. (A) Oskar protein (green) is not present in stage 7 yw egg chambers. Actin is in red. (B) Oskar protein is prematurely translated at the anterior of the oocyte in stage 7 cup ¹/cup⁴⁵⁰⁶ egg chambers. The distribution of Gurken protein (arrows) is normal in both yw (C) and cup ¹/cup⁴⁵⁰⁶ (D) egg chambers. Gurken is green. Actin is red. Bars, 10 μm.

Figure 4.

eIF4E is localized to the posterior pole in a cup-dependent manner. (A) Immunoblot for eIF4E of immunoprecipitates from GFP-Exu extract using α-GFP (GFP), α-Yps (YPS), or rabbit IgG (IgG) antibodies. (B) eIF4E protein (arrows) is concentrated at the posterior of the developing oocyte in stages 1–6 (stage 6 is shown). (C) eIF4E transiently accumulates at the anterior of the oocyte during stages 7 and 8 (late stage 8 is shown). (D) eIF4E then accumulates at the posterior pole during stages 9 and 10 (stage 10 is shown). (E) In yw egg chambers, eIF4E protein (arrow) is concentrated at the posterior of the oocyte (stage 6 is shown). (F) In cup ¹/cup⁴⁵⁰⁶ egg chambers, eIF4E is distrubuted homogenously thoughout the oocyte and nurse cells and is not localized to the posterior pole (arrows; stages 4 and 6 are shown). (G) Doubly transformed yeast expressing a GAL4 DNA binding domain Cup fusion in combination with either a transcriptional activation domain (AD) fusion to eIF4E or the activation domain alone. All interactions were scored based on growth on his^- ade^- media. (H) Deletion analysis of Cup to identify regions required for eIF4E binding. The yellow box is the region of homology with 4E-T, a mouse eIF4E binding protein. The red box is the site of the canonical eIF4E binding motif YXXXXLφ, where X is any amino acid and φ is any hydrophobic amino acid. Bars, 10 μm.

Figure 5.

A model for coupling oskar mRNA localization and translational activation via Cup. (A) During stages 1–7, Cup is required to recruit plus end–directed transport factors, such as Btz. (B) During stages 8 and 9, the oskar RNP rearranges so that Btz can recruit kinesin. (C) During stages 9 and 10, oskar mRNA localizes to the posterior pole and is anchored there. This anchoring event or a posterior localized signal acts on Cup to cause partial disassembly of the complex and breaks the interaction between Cup and eIF4E allowing translation. Asterisks (*) mark interactions that may not be direct.