eIF4A promotes Dpp signalling-dependent Mad and Medea degradation. (a–b) eIF4A induces Tkv^QD-dependent degradation of Mad and Medea. (a) 293T cells were transfected with Flag–Mad, Myc–Medea and eIF4A^WT (or eIF4A^R321H) with or without low-level Tkv^QD (0.5 μg) in combinations, as indicated. Cell lysates were subject to SDS–PAGE and were blotted with anti-Flag, anti-Myc and anti-eIF4A consecutively. Note the apparent degradation of Medea, Mad and eIF4A^WT in the presence of Tkv^QD (lane 6), whereas no degradation was detected in the absence of Tkv^QD (lanes 2–4) or eIF4A^WT (lanes 5 and 7). (b) eIF4A-stimulated degradation depends on the proteosome. 293T cells were co-transfected with Flag–Mad, Myc–Medea, eIF4A^WT (or eIF4A^R321H), and different amounts of Tkv^QD, and treated with the proteosome inhibitor MG-132, as indicated. Note the degradation of Medea and Mad in the presence of eIF4A^WT and, to a lesser extent, eIF4A^R321H at indicated Tkv^QD concentrations, which was inhibited by MG-132. (c) eIF4A promotes Tkv^QD-dependent ubiquitination of Mad. 293T cells were transfected with His–eIF4A^WT, Flag–Mad, Myc–Medea and HA–ubiquitin with or without low levels of Tkv^QD in the presence of MG-132. Mad was immunoprecipitated with anti-Flag and its ubiquitination was detected by anti-HA. Note the increased Mad ubiquitination in the presence of eIF4A (lane 4, compare with lane 2). (d) Ubiquitination of eIF4A depends on the presence of Mad, Medea and Tkv^QD. 293T cells were transfected, as indicated, without added MG-132. eIF4A was immunoprecipitated by anti-histidine and its ubiquitination was detected by anti-HA. Note the dramatic increase of ubiquitinated eIF4A^WT in the presence of Mad, Medea and Tkv^QD (lane 4), compared with either lack of Mad and Medea (lane 2) or Tkv^QD (lane 3). See Supplementary Information Fig. S5 for full-length gels.

Physical association of eIF4A with Mad and Medea. (a) Flag–Mad, Myc–Medea, and eIF4A^WT or eIF4A^R321H were co-transfected into 293T cells with or without Tkv^QD. Cells were stained with anti-Flag (green), anti-eIF4A (magenta) and propidium iodide (PI; red) to reveal the subcellular localization of transfected Mad and eIF4A. (b) 293T cells were co-transfected with Flag–Mad, Myc–Medea, eIF4A^WT and eIF4A^R321H in different combinations, as indicated. Cell lysates were immunoprecipitated (IP) with anti-His (for eIF4A), and the immunoprecipitates were subjected to SDS–PAGE and blotted with antibodies, as indicated. Note that co-immunoprecipitation of Mad and Medea with eIF4A occurred. (c) Drosophila embryo extracts from wild-type or eIF4A^R321H heterozygotes were immunoprecipitated with rabbit anti-eIF4A or goat anti-Mad, or with rabbit anti-βgal or goat anti-HA antibodies (negative controls, respectively), and the immunoprecipitates were subject to SDS–PAGE and blotted with indicated antibodies. (d) As the R321H mutation is located in the carboxy-terminal half of the eIF4A protein¹, which consists of substrate (RNA)-binding domains, the protein was divided into amino- and C-terminal halves, such that eIF4A^WT and eIF4A^R321H shared the same N-terminal fragment but different C-terminal fragments, with a different amino acid at position 321. His-tagged N-terminal, C-terminal, or full-length eIF4A^WT or eIF4A^R321H were expressed from Escherichia coli. The proteins were incubated with Drosophila embryo extracts and were then pulled down by Ni-NTA agarose beads, subjected to SDS–PAGE, and blotted with anti-Mad and anti-histidine. Note that Mad is pulled down by all species of eIF4A proteins, but in higher amounts by eIF4A^WT full-length (lane 6) and C terminus (lane 4). The anti-histidine antibody also recognizes a non-specific band that co-migrated with full-length eIF4A. For each binding assay above, the membrane was blotted with an antibody against the target protein of interest, and was then stripped of bound antibodies and re-blotted with an antibody against the bait protein. See Supplementary Information Fig. S5 for full-length gels.

eIF4A acts synergistally with and independently of DSmurf. (a) eIF4A mutations, but not those of eIF4E or activated TKV, interact synergistically with DSmurf mutations. The effects of maternal mutations on the viability of 1xdpp⁺ versus 3xdpp⁺ progeny are plotted as the ratios of these two categories. Females of the indicated genotypes were mated to 0xdpp⁺/2xdpp⁺ males. The F1 progeny would inherit either 1xdpp⁺ or 3xdpp⁺. Note the ratio is infinity (∞) for progeny of eIF4A¹⁰⁰⁶/+;lack^KG07014/Df mothers, and no progeny were produced by eIF4A^R321H/+;lack^KG07014/Df mothers. Df = Df(2R)Exel7149, which removes DSmurf. (b). eIF4A mutations further enhance the phenotypes of flies lacking maternal and zygotic DSmurf. The cuticles of embryos derived from females of the indicated genotype mated to lack^KG07014/Df males. Dorsalization was quantified by the number of denticles in the first row of the 6^th ventral denticle belt (arrow). Note that embryos heterozygous for eIF4A¹⁰⁰⁶ or eIF4A^R321H and maternally and zygotically null for DSmurf exhibited narrower or no ventral denticle belts, respectively. Inset, high magnification of boxed area showing dorsal hairs. eIF4A^R321H/+;lack/Df females are sterile. They lay very few eggs, most of which seem to be unfertilized (white eggs with no cuticles), regardless to which males they were mated. However, a small percentage (<5%) of the eggs appeared to be fertilized and secreted cuticles. These eggs are completely dorsalized (bottom panel). (c). Mutations in eIF4A further increase Mad protein accumulation in lack/Df embryos. Stage 13 embryos of the indicated genotypes were stained by anti-Mad (dark stains). Note that lack/Df embryos exhibited elevated levels of Mad protein compared with the wild type, similar to eIF4A^R321H/+ embryos (compare with Fig. 1e), and eIF4A¹⁰⁰⁶/+;lack/Df embryos accumulate Mad protein in even more cells, such as in the germ bands and guts (arrows). Right panels are higher magnifications of the boxed area (ps7). Scale bar, 100 μm.

Levels of eIF4A affect Dpp signalling and Mad protein levels. (a) Adult wings of different genotypes are shown anterior side up. Note the ‘thick veins’ (arrows) in the tkv¹/tkv⁴ wing and the suppression by eIF4A¹⁰⁰⁶ or eIF4A^R321H. (b–e) Whole-mount embryos stained with anti-pMad (brown in b, green in c), dpp antisense RNA (blue in d) and anti-Mad (brown in e) are shown anterior to the left and dorsal side up. (b) Higher levels of pMad signals in the dorsal epidermal cells (arrowhead) and posterior midgut (arrow) of stage 10 eIF4A^R321H embryos (right) than in wild-type (WT) embryos (left). (c) At stage 13, pMad signals were detected in parasegment 7 (ps7), gastric caeca (gc) and the epidermis (epd) wild-type embryos (left). In eIF4A^R321H embryos, pMad signals were detected at higher levels and expanded domains in all these tissues, such that pMad staining was seen in significantly larger areas of in gc and ps7 and broader spatial extent in the epidermis (right). (d) Increased dpp transcription is indicated by wider domains of dpp mRNA signals in gc and ps7 in eIF4A^R321H embryos (right). (e) Little or no Mad protein is detected in ps7 of wild-type embryos (left), but is detected in discrete cells of ps7 of eIF4A^R321H embryos (right). (f–g) Adult presumptive gut tissues of the 3^rd instar larvae were doubly stained for GFP (green; marks nuclei of eIF4A⁺ cells), and pMad (magenta in f) or Mad (red in g) are shown partially. Note that increased levels of pMad and Mad were detected within eIF4A¹⁰⁶⁹ mutant cells (lack of green nuclei). Arrows point to nuclei lacking GFP. (h) Expressing dpp by GMR–Gal4 resulted in rough and bulging eyes (left), which was completely suppressed by coexpressing eIF4A⁺ (centre). Expressing dpp by GMR–Gal4 in a eIF4A^R321H/+ background resulted in lethality (4% viability; n = 188). A small number of survivors exhibited more severe roughness and outgrowth of eye tissues (right). (i) Expressing dominant-negative Put or eIF4A by the wing-margin-specific C96–Gal4. (j) Loss of pMad in wing-margin primordial cells (arrow) of the 3^rd instar imaginal discs stained with anti-pMad (green). All scale bars equal 100 μm.