Drosophila Cup is an eIF4E-binding protein that functions in Smaug-mediated translational repression

Abstract

Translational regulation plays an essential role in development and often involves factors that interact with sequences in the 3' untranslated region (UTR) of specific mRNAs. For example, Nanos protein at the posterior of the Drosophila embryo directs posterior development, and this localization requires selective translation of posteriorly localized nanos mRNA. Spatial regulation of nanos translation requires Smaug protein bound to the nanos 3' UTR, which represses the translation of unlocalized nanos transcripts. While the function of 3' UTR-bound translational regulators is, in general, poorly understood, they presumably interact with the basic translation machinery. Here we demonstrate that Smaug interacts with the Cup protein and that Cup is an eIF4E-binding protein that blocks the binding of eIF4G to eIF4E. Cup mediates an indirect interaction between Smaug and eIF4E, and Smaug function in vivo requires Cup. Thus, Smaug represses translation via a Cup-dependent block in eIF4G recruitment.

Figure 1

Cup interacts with Smg and eIF4E. (A) Embryo extracts were mixed with beads carrying the indicated GST fusion protein covalently bound to the resin. Bound proteins were resolved via SDS–PAGE and stained with silver. The position of Cup is indicated. (B) ³⁵S-methionine-labeled Cup (load) generated via in vitro translation was mixed with various GST fusion proteins in the presence of glutathione agarose. Equivalent amounts of load and eluates from the indicated GST fusion proteins are shown. (C) Eluates from (B) were stained with Coomassie blue to detect the captured GST fusion proteins.

Figure 2

Identification of two eIF4E-binding sites in Cup. (A–C) Labeled eIF4E and eIF4E-W106A were generated via coupled transcription/translation in rabbit reticulocyte lysate in the presence of ³⁵S-methionine and mixed with GST fusion proteins carrying various fragments of Cup in the presence of glutathione agarose. Equivalent amounts of the in vitro-translated eIF4E or eIF4E-W106A (load) and the elutions from the indicated GST-Cup proteins are shown. A fraction of each eluate was stained with Coomassie blue to detect the captured GST fusion proteins (GST-Cup). (D) Schematic representation of Cup amino acids 285–487. The ability of various Cup fragments from this region to interact with eIF4E is indicated. Proteins that interact with eIF4E capture 15–47% of the input eIF4E, while fragments that do not interact capture less than 0.5% of the input. These experiments identified two Cup-binding sites: one located within amino acids 335–369 (outlined in gray), which contains a sequence matching the consensus for an eIF4E-binding motif from amino acids 342–348, and a second site from 373–398 (outlined in gray), which does not contain a sequence matching the consensus. Note that the Y342A change within the consensus site and the L379A/L383A change within the second site block eIF4E binding. In addition, the W106A mutation in eIF4E blocks the interaction of the consensus eIF4E-binding site with eIF4E while having no effect on the interaction with the second site.

Figure 3

Cup contains two eIF4E-binding sites. (A) Drosophila S2 cells were transfected with plasmids expressing protein A-tagged wild-type and mutant forms of Cup. Extracts prepared from transfected cells were mixed with ⁷m-GTP-sepharose with and without soluble ⁷m-GDP (cap). Western blots to detect the indicated Cup proteins in the total cell extracts, which represent 15% of protein used in the immunoprecipitations, as well as Cup and eIF4E in the eluates from captures are shown. (B) Labeled eIF4E or eIF4E-W106A as well as wild-type and mutant FLAG-tagged Cup proteins were generated via coupled transcription/translation in rabbit reticulocyte lysate in the presence of ³⁵S-methionine. The eIF4E or eIF4E-W106A was mixed with each of the Cup proteins and immunoprecipitated using an anti-FLAG antibody and protein G agarose. A sample of the in vitro-translated eIF4E (load) representing 50% of protein used in the captures and the elutions from the immunoprecipitations of the indicated Cup proteins are shown.

Figure 4

Cup mediates an indirect interaction between Smg and eIF4E. Labeled eIF4E as well as wild-type and mutant Cup proteins were generated via coupled transcription/translation in rabbit reticulocyte lysate in the presence of ³⁵S-methionine. The eIF4E was mixed with either of the Cup proteins in the presence of GST-Smg^583–763 and glutathione agarose. A sample of the in vitro-translated eIF4E (load) representing 30% of protein used in the captures and the elutions from the captures in the presence of wild-type Cup or Cup^{Y342A/L379A/L383A} are shown.

Figure 5

Co-immunoprecipitation of Smg, Cup, and eIF4E. Extracts derived from embryos collected 0–3 h post-egg-laying were immunoprecipitated with an anti-Smg antibody, an anti-Cup antibody, or normal rat serum in the presence of protein G agarose. Western blots to assay for the indicated proteins in crude embryo extracts as well as the indicated immunoprecipitates are shown. Where indicated, immunoprecipitations were performed in the presence of RNase A or employing extracts derived from embryos collected from smg mutant mothers. Embryo extract lanes represent 5% of the material used in the immunoprecipitations.

Figure 6

Cup's eIF4E-binding sites block the eIF4E/eIF4G interaction. A labeled fragment of Drosophila eIF4G (residues 434–804), which carries the protein's eIF4E-binding motif, was generated via coupled transcription/translation in rabbit reticulocyte lysate in the presence of ³⁵S-methionine and mixed with a resin carrying covalently coupled eIF4E. Captures were performed in the presence of increasing amounts of either GST-Cup^335–359 or GST-Cup^{335–359Y342A}, which correspond to the wild-type and mutant version of the Cup eIF4E-binding motif, respectively, or GST-Cup^361–410 or GST-Cup^{361–410L379A/L383A}, which correspond to the wild-type and mutant version of the Cup second eIF4E-binding site, respectively, or a wild-type or mutant version of a 20-mer peptide corresponding to the eIF4G protein's eIF4E-binding motif. The effect of these competitors on eIF4G capture was quantitated and expressed as the fraction of eIF4G captured in the absence of competitors.

Figure 7

Cup functions in Smg-mediated translational repression. (A,B) Embryos derived from mothers with the indicated genotypes were injected with both Renilla luciferase and luc3 × SRE+ RNAs, or Renilla luciferase and luc3 × SRE− RNAs. Injected embryos were aged, and firefly and Renilla enzyme levels were assayed in embryo extracts. After correcting the levels of firefly enzyme activity using the levels of Renilla activity as a control, the amount of Smg-mediated repression (fold repression) was quantitated by dividing the corrected luc3 × SRE− value for a given genotype by the corresponding corrected luc3 × SRE+ value for that genotype. (C) Ovaries and embryos from mothers with the indicated genotypes were assayed for the levels of Cup and β-tubulin via Western blot. The levels of Cup protein in embryos were quantitated using β-tubulin as a loading control, and the amount of Cup in each sample relative to wild type is indicated.