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First published online May 16, 2007
doi: 10.1242/10.1242/dev.001594

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Dissection of the target specificity of the RNA-binding protein HOW reveals dpp mRNA as a novel HOW target

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Characterization of the HOW response element. Western blot analysis of HA-tagged HOW following precipitation with distinct biotin-labeled RNA fragments. (A) Top, schematic representation of the putative HOW response element (HRE) sites in the stripe 3'UTR. Bottom, the sequences of the HRE sites in the RNA samples (at 0.4 µM concentration) are indicated below the blot; wild type (ACUAA, left) or with point mutations in the HRE site (AC, middle or AG, right). HOWm was used as a control for non-specific binding because it mimics the howe44 allele, which does not bind RNA. (B) The wild-type stripe 3'UTR (0.4 µM) (two right-hand lanes) or the stripe 3'UTR with point mutations in all four HRE sites (two left-hand lanes). (C) Binding of HOW to RNA fragments (0.4 µM) representing 0-533 nucleotides of the stripe 3'UTR (0 represents the last nucleotide of the stop codon), containing a single HRE site (1), or representing nucleotides 0-610, containing three HRE sites (3). (D) HRE in loop structures of variable sizes (loop sizes are indicated) fused to the 1-225 nucleotide (nt) RNA fragment of stripe 3'UTR (0.4 µM). (E) Biotin-labeled RNA oligomers containing a single HRE within the distinct secondary-structure motifs; unstructured, loop, stem, stem and loop junction 1, and stem and loop junction 2 (illustrated below) at concentrations of 0.2 µM (1), 0.02 µM (2), 0.002 µM (3). *Indicates the HRE.

View larger version (43K): [in this window] [in a new window]   Fig. 2. Large howstru mutant clones exhibit broadening of the Spalt, the DPP direct target, domain. Wild-type wing imaginal discs (A-D) or wing imaginal disc with large howstru mutant clones (E-H) stained for HOW (red; A,E), Spalt (green; B,F) and Engrailed (blue; C,G). (D,H) Merged images of HOW (red) and Spalt (green) expression. Arrowheads indicate the anteroposterior boundary. (I) Quantification of the Spalt (Sal) domain width in wild-type (WT) or in mutant (CLONES) discs is given as the ratio between a cumulative value representing Spalt width (in upper, lower and middle regions) and the wing imaginal disc width (Sal/disc).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Two HRE sites in the dpp 3'UTR mediate the binding and repression of HOW. (A) Schematic representation of the two HRE sites in the 3'UTR of dpp. These sites were mutated as described. (*) Wild-type sequences; () mutated sequence. (B) Western blot analysis for HA-tagged HOW or HOWm (HOW-HA) following precipitation with the wild-type (WT) or mutated dpp 3'UTR. (C) Western blot analysis with anti-GFP, anti-actin and anti-TAP of S-2R+ cells transfected with GFP-dpp3'UTR containing wild-type or mutated HRE sites, together with or in the absence of HOW(L). The bar graph represents the results of three independent transfection experiments that were normalized to the actin levels of each sample.

View larger version (92K): [in this window] [in a new window]   Fig. 4. HOW(L) represses the levels of DPP-GFP in a 3'UTR-dependent manner. (A-L) Wing imaginal discs labeled with GFP (A,D,G,J) or with anti-HOW (red; B,E,H,K) were dissected from larvae carrying the vestigial-gal4 driver and UAS-HOW(L) (D-F,J-L), and either a dpp-GFP fusion construct that contains the dpp3'UTR (A-F) or one lacking the dpp 3'UTR (G-L). The corresponding merged panels are shown (C,F,I,L). Arrows show reduced GFP expression in the presence of HOW(L) and dpp3'UTR, and normal GFP expression in the absence of the 3'UTR of dpp. Note a significant reduction of DPP-GFP in the presence of HOW(L) only when the dpp-GFP construct contains the endogenous dpp 3'UTR (D).

View larger version (97K): [in this window] [in a new window]   Fig. 5. Overexpression of HOW(L) reduces the Spalt domain non-autonomously and represses dpp mRNA expression. (A-H) Wing imaginal discs carrying sd-gal4 (A-D) or dpp-gal4 (E-H) and either UAS-GFP alone (A,B,E,F) or together with the repressor isoform UAS-HOW(L) (C,D,G,H) stained for Spalt (Sal, red). The merged images of GFP and Spalt are shown (B,D,F,H). (I) Quantification of the Spalt domain width in wild type (WT) and in wing imaginal discs overexpressing HOW(L) in the dpp domain is given as the ratio between Spalt domain width and the wing imaginal disc width (calculated as detailed in Fig. 2). (J,K) In situ hybridization with the DIG-labeled dpp antisense probe of wing imaginal discs carrying sd-gal4 (J), or overexpressing HOW(L) driven by the sd-gal4 driver (K). Arrowheads indicate the wing imaginal disc pouch domain.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Reduction of HOW levels elevates endogenous dpp mRNA. (A-C) In situ hybridization of wing imaginal discs with a dpp-specific probe carrying the sd-gal4 driver alone (A), or together with how-specific dsRNA (B) or with how(L)-specific dsRNA (C). Arrowheads indicate the sd-expression domain. Notice the specific elevation of dpp in the wing imaginal disc pouch in B and C. (D-I) Wing imaginal discs carrying the sd-gal driver and UAS-GFP together with how-specific dsRNA (D,E,F) or together with how(L)-specific dsRNA (G,H,I) stained for anti-HOW (D,G) and labeled with GFP (E,H). Their corresponding merged images are shown in F and I. Notice the specific reduction of HOW in the sd-gal4 expression domain in both D and G. (J-L) Adult wings of flies carrying the sd-gal4 driver alone (J), or in combination with how-specific dsRNA (K) or with how(L)-specific dsRNA (L). Arrows point to wing blisters. (M,N) Adult wings of flies carrying the ms1096-gal4 alone (M) or together with how(L)-specific dsRNA (N). Arrow points to extra veins.