Cup is an eIF4E binding protein required for both the translational re



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 25 下一页

返回列表

查看: 390960\|回复: 241	go Cup is an eIF4E binding protein required for both the translational re

杨柳

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 3465

发表于 2009-3-6 00:56 |显示全部帖子

1 Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 212104 C4 w# M5 i4 y: ?5 D

2 Genentech, Inc., South San Francisco, CA 94080

Address correspondence to James Wilhelm, Department of Embryology, Carnegie Institution of Washington, 115 West University Parkway, Baltimore, MD 21210. Tel.: (410) 554-8192. Fax: (410) 243-6311. email: wilhelm@ciwemb.edu

Abstract8 \% d1 E9 l; C, j; [, p4 [/ ]

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.

Key Words: oskar mRNA; oogenesis; Drosophila; Barentsz; eIF4E binding protein; {2 P! ^+ ]6 b, n# V: X! n
: B( T  L  ?1 t6 @! \, e* _. o
The online version of this article includes supplemental material.

Abbreviations used in this paper: Btz, Barentsz; eIF4E, eukaryotic initiation factor 4E; Exu, Exuperantia; Yps, Ypsilon Schachtel.

Introduction
* E1 X3 S8 Q  B
Localization of mRNAs is used by many polarized cells as a means of restricting the distribution of a protein to a particular cytoplasmic domain. One of the most extensively characterized systems for studying mRNA localization is the Drosophila oocyte (Bashirullah et al., 1998; Johnstone and Lasko, 2001). The basic unit of Drosophila oogenesis is the egg chamber, which is comprised of an oocyte and 15 nurse cells surrounded by a layer of somatic follicle cells. The oocyte is connected to the nurse cells by a network of cytoplasmic bridges called ring canals. This network allows the nurse cells to synthesize various mRNAs that are required for early embryogenesis and transport them in a microtubule-dependent manner to discrete locations within the oocyte (Pokrywka and Stephenson, 1995). The correct localization of oskar mRNA to the posterior pole is particularly crucial for development since this localization is essential for both posterior patterning and establishment of the germ line (Ephrussi et al., 1991). During early oogenesis (stages 1–6), oskar mRNA accumulates at the posterior pole of the oocyte where the minus ends of the microtubule array are concentrated (Fig. 1 A) (Ephrussi et al., 1991; Kim-Ha et al., 1991; Theurkauf et al., 1993). At stages 7 and 8, the microtubules reorganize so that microtubule nucleation occurs over most of the oocyte cortex with the majority of the minus ends being concentrated at the anterior of the oocyte (Fig. 1 A) (Cha et al., 2002). Tracking the minus ends of the microtubules, oskar mRNA transiently localizes to the anterior of the oocyte during these stages (Ephrussi et al., 1991; Kim-Ha et al., 1991). During stages 9 and 10, however, oskar mRNA transits back to the posterior pole in a plus end–directed transport step that requires kinesin heavy chain (khc) (Fig. 1 A) (Brendza et al., 2000). Once oskar mRNA reaches the posterior pole it is translated (Fig. 1 A). The mechanism for coupling translational activation to completion of the last step in oskar mRNA localization has remained elusive.. H0 Z$ D$ s2 w
5 T& D7 s9 `3 o4 {: ]+ C* i
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 -GFP (GFP), -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) -Cup staining. (G) -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) -Cup staining. (J) -Btz staining. (H) Merged image. Cup is in red and Btz is in green. Bars, 10 μm.4 k! U: o/ Q! q2 O
( |+ ?$ P: U/ l, e- g$ W8 h. G
One general model for how coupling of localization and translation might occur is that there are factors common to both the localization and translational control complexes that are required to coordinate the completion of localization with translational activation. Mutations in a gene product that is common to both complexes might be predicted to cause mislocalization of oskar mRNA and premature translation of the oskar message. However, mutants that disrupt oskar mRNA localization typically have phenotypes similar to those observed in barentsz (btz) mutants: failure of plus end–directed transport of the oskar message during stages 9 and 10, resulting in a complete lack of oskar translation (van Eeden et al., 2001). Conversely, a number of translational repressors of oskar mRNA (e.g., BicC, bruno, ME31B) have been identified, but their effects on oskar mRNA localization appear to be limited (Kim-Ha et al., 1995; Saffman et al., 1998; Nakamura et al., 2001). For instance, mutating all of the Bruno response elements in the oskar 3'UTR causes premature translation of oskar at stages 7 and 8, but does not interfere with oskar mRNA localization (Kim-Ha et al., 1995). Thus, although a number of components are known to be required for either localization or translational repression, no component isolated to date appears to be a part of both complexes./ A1 g2 s, V1 j6 Y8 L' t. Z. p
  o  ?$ E. j0 U
To identify new components of the oskar RNP complex, we previously purified an eight-protein complex that contains oskar mRNA (Wilhelm et al., 2000). In this study, we identify the 147-kD protein of this complex as the product of the female sterile gene cup. Surprisingly, cup is required both for translational repression and localization of oskar mRNA. We also demonstrate that Cup binds to eukaryotic initiation factor 4E (eIF4E) and is necessary to recruit the localization factor Barentsz to the complex. Thus, Cup is a translational repressor of oskar that is required to assemble the oskar mRNA localization machinery. Because of its interactions with both the localization and translational control complexes, we propose that Cup is a likely regulatory target for the coupling machinery.0 l" b' p9 N. [% n. v" }7 q& e  g1 r
3 Z3 x; ~" q, J2 ?! Y$ r
Results and discussion

Cup is a component of the oskar RNP complex
4 i! c( e- H' n1 y3 r: |
To identify novel components that play a role in either localization or translational regulation of oskar mRNA, we previously purified an oskar RNP complex that contains Exuperantia (Exu), Ypsilon Schachtel (Yps), and six unidentified proteins (Wilhelm et al., 2000). Using mass spectrometry, we identified the 147-kD protein of this complex as Cup. To confirm that Cup is a bona fide component of the oskar RNP complex, we immunoprecipitated both GFP-Exu and Yps and immunoblotted with -Cup antibody. Cup specifically coimmunoprecipitates with both GFP-Exu and Yps, demonstrating that Cup is a component of the complex (Fig. 1 B).
, Q) M) \: z0 w; ?& I5 y0 U
cup was originally identified as a female sterile mutation that forms eggs that are open at the anterior due to a failure in chorion deposition at the anterior of the oocyte (Schupbach and Wieschaus, 1991; Keyes and Spradling, 1997). This previous work established that Cup is a cytoplasmic protein that is localized early to the oocyte (Keyes and Spradling, 1997). Since Cup copurifies with components of an oskar RNP complex, we decided to examine the distribution of Cup during oogenesis in more detail. Immunostaining of different stage egg chambers (see Spradling, 1993, for staging) revealed that Cup accumulates at the posterior of the oocyte during stages 1–6, consistent with previously published results (Fig. 1 C) (Keyes and Spradling, 1997). At stages 7 and 8, Cup was localized to the anterior of the oocyte (Fig. 1 D), followed by redistribution to the posterior of the oocyte during stages 9 and 10 (Fig. 1 E). Thus, Cup copurifies with components of the oskar RNP complex and is localized within the oocyte in a temporal–spatial pattern identical to that of oskar mRNA.

One of the rationales for using GFP-Exu as a biochemical handle for the purification of localization complexes is that GFP-Exu forms particles in nurse cells that move in a microtubule-dependent manner (Theurkauf and Hazelrigg, 1998). Previously, we demonstrated that Yps, which binds directly to Exu, localizes to these motile particles (Wilhelm et al., 2000). To determine if Cup is also a component of these particles, we immunostained egg chambers for both Cup and Yps. The particulate staining observed for both Cup and Yps in the nurse cells showed a high degree of overlap, indicating that Yps and Cup are part of the same particles in vivo (Fig. 1, F–H). Recently, a novel component of the oskar mRNA localization machinery, Btz, was identified that has a staining pattern that is strikingly similar to that of Cup (van Eeden et al., 2001). We immunostained egg chambers for both Cup and Btz to determine if they were also present in the same nurse cell particles. Most cytoplasmic particles contained both Cup and Btz (Fig. 1, I–K). Interestingly, Btz protein that localized tightly to the nuclear rim did not display a large amount of overlap with Cup (Fig. 1 K), indicating that this pool of Btz might be part of a separate complex. Thus, Cup is present in motile RNP particles that contain Btz, a known component of the oskar mRNA localization machinery.

Cup is required for oskar mRNA localization.

Since Cup colocalizes and copurifies with components of the oskar RNP complex, we next asked if Cup plays a role in oskar mRNA localization. For this and subsequent experiments, we focused our attention on the heteroallelic combination of cup1/cup4506 since the combination of the strong cup4506 allele with the intermediate strength cup1 allele allowed oogenesis to proceed far enough to assay oskar mRNA localization. This allelic combination yielded results that were representative of other heteroallelic combinations (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200309088/DC1) and also allowed us to minimize the effects of secondary mutations since cup1 and cup4506 were isolated in separate screens. In situ hybridization of oskar mRNA in cup1/cup4506 egg chambers revealed that although oskar mRNA localization is normal in stages 1–7 of oogenesis (Fig. 2, A and B, D and E), during stages 8- 10, oskar mRNA is predominantly cortical with some enrichment at the posterior pole (Fig. 2, C and F). This dispersed localization pattern is similar to that observed in weak alleles of btz where low levels of oskar mRNA are localized to the posterior pole (van Eeden et al., 2001).
+ B3 r# |) B  i7 {- Y0 K. \% \- y
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 cup1/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 cup1/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 cup1/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 cup1/cup4506 (J) egg chambers. Actin is in red. Bars, 10 μm.  `2 J% X9 S) f4 L' R
/ ]$ D. ~9 q( @. D/ a' e
Cup is required to recruit the localization factor Btz

Because btz mutants display a late stage oskar mRNA localization defect similar to that of cup mutants (van Eeden et al., 2001), we next examined the effect of cup mutants on the distribution of Btz. Normally, Btz protein is present on the nuclear envelope in nurse cells and colocalizes with oskar mRNA in the oocyte (Fig. 2 G). However, in cup1/cup4506 egg chambers, the accumulation of Btz protein within in the oocyte is greatly reduced from stage 1 onward, whereas the Btz present on the nuclear envelope in the nurse cells is unaffected (Fig. 2 H). The failure in the transport of Btz to the oocyte is not due to a general defect in assembly of the oskar RNP since cup1/cup4506 egg chambers localize Yps and oskar mRNA normally during early oogenesis (Fig. 2, I and J, D and E; Figs. S1 and S2, available at http://www.jcb.org/cgi/content/full/jcb.200309088/DC1). Thus, Cup is specifically required to localize Btz to the oocyte. This result, together with the findings that Cup and Btz colocalize as well as sharing similar oskar mRNA localization defects, argues that cup mutants fail to localize oskar mRNA because Cup is required to recruit Btz to the complex./ {% {3 R) Y. F% S5 B4 i$ v5 O

Cup is required to maintain translational repression of oskar mRNA
, z5 j+ \/ h; L5 B! J+ y) ^
Since all mutations isolated to date that disrupt oskar mRNA localization also block oskar translation, we next examined the role of cup in oskar translation. To our surprise, Oskar protein accumulated prematurely in the oocyte during stages 6 and 7 in cup1/cup4506 egg chambers, indicating that cup is required to translationally repress oskar mRNA during these stages (Fig. 3, A and B; Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200309088/DC1). It is also worth noting that in cup mutants we only observe accumulation of Oskar protein at those sites where oskar mRNA is most enriched (Fig. 3 B; Fig. S3). This may be due to the fact that the cup alleles used in this study are hypomorphic alleles. The effects of cup are specific for oskar mRNA since the localized translation of gurken mRNA at the dorsal anterior region of the oocyte during stage 9 is unaffected in a cup1/cup4506 mutant background (Fig. 3, C and D). Thus, cup is not a general translational regulator of localized messages.0 N4 Q$ Q: m- [5 ]( o; I' b: M

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 cup1/cup4506 egg chambers. The distribution of Gurken protein (arrows) is normal in both yw (C) and cup1/cup4506 (D) egg chambers. Gurken is green. Actin is red. Bars, 10 μm.
3 H& a9 X0 B; V
eIF4E is localized to the posterior pole in a cup-dependent manner3 T0 R  r8 K: a; i0 U3 B" P& N

To better understand the role of Cup in maintaining the translational repression of oskar mRNA, we first sought to identify components of the translation machinery that were present in the complex by testing likely candidates. Immunoprecipitation of GFP-Exu and Yps showed that eIF4E, the 5' cap binding component of the translation initiation complex, is specifically associated with these components of the oskar RNP complex (Fig. 4 A). eIF4E and other components of the translation initiation machinery are generally thought of as being homogenously distributed due to their critical role in translation throughout the cell. Surprisingly, we found that eIF4E is localized in a dynamic pattern within the oocyte. eIF4E is localized to the posterior of the oocyte early in oogenesis during stages 1–6 (Fig. 4 B). At stages 7 and 8, eIF4E redistributed to the anterior of the oocyte (Fig. 4 C), and during stages 9 and 10, eIF4E accumulated at the posterior of the oocyte (Fig. 4 D). This pattern of localization was also observed with a GFP-eIF4E protein trap line (unpublished data). Thus, eIF4E localizes in a temporal–spatial pattern identical to that of Cup, suggesting that it is a component of the complex in vivo.
3 @, v- _) G1 b7 R* ~
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 -GFP (GFP), -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 cup1/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 YXXXXL, where X is any amino acid and  is any hydrophobic amino acid. Bars, 10 μm.

Since Cup is required for the correct localization of Btz to the oocyte, we next investigated whether Cup is required for eIF4E localization. Immunostaining of cup1/cup4506 mutant egg chambers revealed that Cup is required for localization of eIF4E to the posterior of the oocyte from stage 1 onward (Fig. 4, E and F). Disruption of cup function did not significantly affect the level of unlocalized eIF4E (Fig. 4, E and F), indicating that the defect is primarily in the recruitment of eIF4E to the complex.
4 Y0 d/ @+ ]6 h0 |: R6 h6 j
Because Cup shares limited homology with 4E-T, a known eIF4E binding protein and a translational repressor in mammals (Dostie et al., 2000), we tested whether Cup binds to eIF4E using a two-hybrid interaction assay. This assay showed a direct interaction between Cup and eIF4E (Fig. 4 G). Cup interacted equally with both isoforms of eIF4E (unpublished data). Deletion analysis of Cup using the two-hybrid assay identified an eIF4E interaction domain that contains a canonical eIF4E binding motif (Fig. 4 H). This motif is found in eIF4G as well as translational repressors (e.g., 4E-T) that block translation by preventing the eIF4E–eIF4G interaction (Mader et al., 1995). Thus, Cup is an eIF4E binding protein that acts directly to repress oskar translation.( R+ k# a/ H6 ~, w; v2 ]
0 J% l9 M" A# k! T0 e: i
Although mRNA localization in Drosophila has been the subject of extensive genetic analysis, only a few attempts have been made to characterize biochemically the proteins associated with localized messages. In this study, we have biochemically identified Cup as a novel component of the oskar RNP complex. This assignment is based on a number of findings. First, Cup copurifies with both Exu and Yps, which have both been shown to be in a biochemical complex with oskar mRNA. Second, Cup protein exhibits the same dynamic localization pattern as that seen for oskar mRNA as well as other components of the complex. Third, Cup colocalizes with Yps and Btz particles, indicating that this these proteins form a complex in vivo. Finally, the relevance of the biochemical association is supported by genetic studies of cup function, demonstrating a role for cup in translational repression of oskar mRNA as well as recruitment of Btz and eIF4E to the RNP complex.- B% W+ v( n- i) U5 g& |. }
2 a, D4 V3 ]4 D
A model for coupling oskar localization to translational derepression

Because Cup is a translational repressor that is also required to assemble the oskar mRNA localization machinery, we propose that the coupling between localization and translation occurs by regulating these two functions of Cup. In this model, Cup is required early in the assembly of the transport complex in order to recruit components, such as Btz, that will later be used to dock to kinesin (Fig. 5 A). This is consistent with our results that cup is required to localize Btz to the posterior pole and that cup mutants exhibit oskar mRNA localization defects comparable to those observed in btz mutants. The fact that mammalian Btz and 4E-T are nucleocytoplasmic shuttling proteins suggests that the defect in particle assembly in cup mutants may occur in the nucleus rather than in the cytoplasm (Dostie et al., 2000; Macchi et al., 2003). However, further studies will be necessary to determine the site of assembly.
$ p2 P, [' _) r# P2 V
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.

Because Btz is normally part of the transport complex throughout oogenesis even though it is only required for the kinesin-mediated transport step during stages 9 and 10 (van Eeden et al., 2001), we further propose that the complex undergoes rearrangement in order to activate Btz and switch from minus end–directed transport to kinesin-mediated transport (Fig. 5 B). Since we have yet to establish the direct binding of Cup to Btz or Btz to kinesin it is unclear how many components of the complex may be involved in this reorganization.
  F8 i' v# S+ Z; t
Once the complex reaches the posterior pole, we argue that the localization machinery is disassembled and the interaction between Cup and eIF4E is broken to allow translational activation (Fig. 5 C). Because Cup is stably maintained at the posterior pole after stage 9, whereas Btz is not (this study; van Eeden et al., 2001), we propose that the trigger that disrupts the binding of Cup to eIF4E also leads to partial disassembly of the localization machinery via Cup. The molecular trigger for such rearrangements is unknown, however, the ability of 4E-T to bind eIF4E is regulated by phosophorylation (Pyronnet et al., 2001). Studies directed at identifying regulators of the Cup–eIF4E interaction might lead to greater mechanistic insights into the coupling mechanism.

One of the attractive features of this model is that it suggests how coupling might be accomplished in other systems. Recent work in neurons on the translational regulator CPEB suggests that it can promote the transport of mRNA into dendrites (Huang et al., 2003). Since CPEB represses translation by recruiting the eIF4E binding protein, maskin, to transcripts (Stebbins-Boaz et al., 1999), it is possible that the observed transport effect is due to a requirement for maskin to assemble the localization machinery. Thus, Cup may be representative of a general class of eIF4E binding proteins whose role is to couple mRNA localization to translational activation.. k8 y6 a0 e7 h" s
+ O, K, J5 |0 E6 y
Materials and methods1 S. p7 A; @2 h; p% p! j
0 o6 l7 C" I' Q/ F( z8 l& {
Drosophila strains and culture* p& ~' t" H0 P- x  P7 p
3 Y! a6 l0 {! w0 v4 \
Fly stocks were cultured at 22–25°C on standard food. The cup1, cup4, cup1355, and cup4506 alleles have been previously described (Schupbach and Wieschaus, 1991; Keyes and Spradling, 1997). The y1 w67c23 strain is described in FlyBase.

Extract preparation, immunoblots, and immunoprecipitations

All protein work was performed as previously described (Wilhelm et al., 2000). For immunoblot analysis, primary antibodies were used at a 1:1,000 dilution of -Cup rat antibody (Keyes and Spradling, 1997) or 1:1,000 -eIF4E rabbit antibody (a gift from P. Lasko, McGill University, Montréal, Canada).1 W% [+ j) H* w0 C

Identification of p1479 a$ u" |* ~) H
: {, b$ Y# c+ j4 l8 C/ B; Y' x! [) L
p147 was resolved by SDS-PAGE and mass spectrometry performed as described (Wang et al., 1999).
) ?* w" G# a, @: G' O9 ]5 l
Immunostaining and fluorescence microscopy7 Y5 U4 h! |5 k3 {2 e+ i
) }0 J3 b. M8 [5 ]% l9 ~$ h
Immunostaining and microscopy was performed as previously described (Cox and Spradling, 2003) with the following modifications: the washes immediately following fixation consisted of PBT (1 x PBS, 0.2% Triton X-100). All subsequent washes or incubations were done in PBT 5% BSA; primary antibodies were diluted in PBT 5% BSA as follows: rat -Cup 1:1,000 (Keyes and Spradling, 1997), rabbit -Osk 1:3,000 (a gift from A. Ephrussi, European Molecular Biology Laboratory, Heidelberg, Germany), rabbit -Btz 1:1,000 (van Eeden et al., 2001), rabbit -Yps 1:1,000 (Wilhelm et al., 2000), 1:1 mouse -Grk (1D12, Developmental Studies Hybridoma Bank), rabbit -eIF4E 1:1,000 (a gift from P. Lasko). The following secondary antibodies were used: goat -rabbit and -rat AlexaFluor488 (1:200) and goat -rat AlexaFluor568 (1:200). Samples were mounted in Vectashield. Confocal analysis was performed using the PL APO40X 1.25NA and 100X 1.40NA objectives on the Leica TCS NT confocal microscope at 25°C., J) G, L6 t/ X4 T: q8 x# G
1 m: {& `: p+ H$ h
In situ hybridization4 b- ]" }! O+ Z2 k2 c' ?) n
* O% V, t8 K% d/ [* V
In situ hybridization and detection were performed as described (Wilkie et al., 1999).

Two-hybrid analysis of cup and eIF4E* N4 ~0 {  R& I$ L. \" c0 `

The Rf cassette (Invitrogen) was inserted into the two-hybrid vectors, pGADT7 and pGBKT7 (CLONTECH Laboratories, Inc.), to facilitate cloning via the Gateway cloning system (Invitrogen). The following deletion constructs were generated by PCR and were cloned into into the appropriate vector for analysis: CupA 1–912 aa, CupB 1–652 aa, CupC 1–457 aa, CupD 1–233 aa, CupE 233–457 aa. Transformants were tested for positive interactions based on their ability to grow on leu- trp- his- ade- plates as described in the protocols for the Clontech matchmaker system (CLONTECH Laboratories, Inc.). The expression of all constructs was confirmed by immunoblot of yeast lysate with either -myc (9E10) or -HA (12CA5) antibodies.$ k; M& ?/ Z0 b7 q2 h1 C0 `
7 v% ?) a$ E; f8 o0 l0 x/ t5 @: {$ m
Online supplemental material

Online supplemental figures are available at http://www.jcb.org/cgi/content/full/jcb.200309088/DC1. Fig. S1 shows the effect of other heteroallelic combinations of cup on oskar mRNA localization and localization of Btz. Fig. S2 shows the localization of Yps and eIF4E in a variety of stages of cup1/cup45066 egg chambers. Fig. S3 shows oskar derepression in cup1/cup45066 egg chambers during stages 6–9.6 c# k4 f9 O- T. D

Acknowledgments
6 L/ y- O0 h+ U: o+ w0 j
The authors would like to thank D. St. Johnston, A. Ephrussi, P. Lasko, and A. Spradling for strains and antibodies.; f5 r. y8 E1 ]0 e

J. Wilhelm is a Howard Hughes Medical Institute fellow of the Life Sciences Research Foundation.

References4 a& n. s/ Z4 z: h" M
3 H, q+ m4 G& b0 C. z+ J
Bashirullah, A., R.L. Cooperstock, and H.D. Lipshitz. 1998. RNA localization in development. Annu. Rev. Biochem. 67:335–394.
% s0 t0 V. u5 s
Brendza, R.P., L.R. Serbus, J.B. Duffy, and W.M. Saxton. 2000. A function for kinesin I in the posterior transport of oskar mRNA and Staufen protein. Science. 289:2120–2122.
& x/ F) Y  T( m% {( X
Cha, B.J., L.R. Serbus, B.S. Koppetsch, and W.E. Theurkauf. 2002. Kinesin I-dependent cortical exclusion restricts pole plasm to the oocyte posterior. Nat. Cell Biol. 4:592–598.
  y6 C7 v4 e2 c7 ~
Cox, R.T., and A.C. Spradling. 2003. A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development. 130:1579–1590.) V6 J) W  L9 v/ u

Dostie, J., M. Ferraiuolo, A. Pause, S.A. Adam, and N. Sonenberg. 2000. A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E. EMBO J. 19:3142–3156.4 l3 o: o' s. g  g

Ephrussi, A., L.K. Dickinson, and R. Lehmann. 1991. Oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell. 66:37–50.6 y' {+ }: A! `
; w$ P& c, b+ f' z+ A5 M
Huang, Y.S., J.H. Carson, E. Barbarese, and J.D. Richter. 2003. Facilitation of dendritic mRNA transport by CPEB. Genes Dev. 17:638–653.4 y/ G  h. l' w6 f

Johnstone, O., and P. Lasko. 2001. Translational regulation and RNA localization in Drosophila oocytes and embryos. Annu. Rev. Genet. 35:365–406.4 Y# r# j# {$ `7 b$ h" @8 d

Keyes, L.N., and A.C. Spradling. 1997. The Drosophila gene fs(2)cup interacts with otu to define a cytoplasmic pathway required for the structure and function of germ-line chromosomes. Development. 124:1419–1431.) @' }. H4 i7 X5 A# i0 _

Kim-Ha, J., K. Kerr, and P.M. Macdonald. 1995. Translational regulation of oskar mRNA by bruno, an ovarian RNA-binding protein, is essential. Cell. 81:403–412.

Kim-Ha, J., J.L. Smith, and P.M. Macdonald. 1991. oskar mRNA is localized to the posterior pole of the Drosophila oocyte. Cell. 66:23–35.

Macchi, P., S. Kroening, I.M. Palacios, S. Baldassa, B. Grunewald, C. Ambrosino, B. Goetze, A. Lupas, D. St. Johnston, and M. Kiebler. 2003. Barentsz, a new component of the Staufen-containing ribonucleoprotein particles in mammalian cells, interacts with Staufen in an RNA-dependent manner. J. Neurosci. 23:5778–5788.' t- s, _3 V6 k& N/ ~: @4 k
9 j9 R# H" t8 g0 I
Mader, S., H. Lee, A. Pause, and N. Sonenberg. 1995. The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol. Cell. Biol. 15:4990–4997.( F; g8 Z, N# G( S# g# C7 Y! q
8 U+ l# U' p: Z, R
Nakamura, A., R. Amikura, K. Hanyu, and S. Kobayashi. 2001. Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis. Development. 128:3233–3242.9 w! b" |5 J4 F* i

Pokrywka, N.J., and E.C. Stephenson. 1995. Microtubules are a general component of mRNA localization systems in Drosophila oocytes. Dev. Biol. 167:363–370.
" |& n) U, [  F4 s& m* d% a
Pyronnet, S., J. Dostie, and N. Sonenberg. 2001. Suppression of cap-dependent translation in mitosis. Genes Dev. 15:2083–2093.
4 V2 |+ C( X+ X- U; o: X
Saffman, E.E., S. Styhler, K. Rother, W. Li, S. Richard, and P. Lasko. 1998. Premature translation of oskar in oocytes lacking the RNA-binding protein bicaudal-C. Mol. Cell. Biol. 18:4855–4862.

Schupbach, T., and E. Wieschaus. 1991. Female sterile mutations on the second chromosome of Drosophila melanogaster. II. Mutations blocking oogenesis or altering egg morphology. Genetics. 129:1119–1136.0 e! t, x( H9 q
8 n7 G- ?. G9 n5 c
Spradling, A.C. 1993. Developmental genetics of oogenesis. The Development of Drosophila melanogaster. Vol. 1. Cold Spring Harbor Laboratory Press, Plainview, NY. 1–70." A! j* d% q6 ?
" I3 h* L, J: F: G; S0 d+ ~* f
Stebbins-Boaz, B., Q. Cao, C.H. de Moor, R. Mendez, and J.D. Richter. 1999. Maskin is a CPEB-associated factor that transiently interacts with elF-4E. Mol. Cell. 4:1017–1027.

Theurkauf, W.E., B.M. Alberts, Y.N. Jan, and T.A. Jongens. 1993. A central role for microtubules in the differentiation of Drosophila oocytes. Development. 118:1169–1180.

Theurkauf, W.E., and T.I. Hazelrigg. 1998. In vivo analyses of cytoplasmic transport and cytoskeletal organization during Drosophila oogenesis: characterization of a multi-step anterior localization pathway. Development. 125:3655–3666.+ o- ?2 I: Z5 W# p, E* N8 W! l3 N1 b& g+ f

van Eeden, F.J., I.M. Palacios, M. Petronczki, M.J. Weston, and D. St. Johnston. 2001. Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole. J. Cell Biol. 154:511–523.8 o5 t4 A, R8 G- w* ]
4 M2 s) H" q1 B1 a, r. w# m! v8 u9 N. n
Wang, K.H., K. Brose, D. Arnott, T. Kidd, C.S. Goodman, W. Henzel, and M. Tessier-Lavigne. 1999. Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching. Cell. 96:771–784.4 Z! O, v6 {4 a+ d+ |' [5 m
6 f: x! p4 p1 }  f  N% t: b
Wilhelm, J.E., J. Mansfield, N. Hom-Booher, S. Wang, C.W. Turck, T. Hazelrigg, and R.D. Vale. 2000. Isolation of a ribonucleoprotein complex involved in mRNA localization in Drosophila oocytes. J. Cell Biol. 148:427–440.) ?& D! Q# ]3 ^- j+ E% J3 i, N' \0 y
% r9 O. E5 ^% N( c
Wilkie, G.S., A.W. Shermoen, P.H. O'Farrell, and I. Davis. 1999. Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei. Curr. Biol. 9:1263–1266.(James E. Wilhelm1, Meredith Hilton1, Qui)