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First published online 28 November 2007
doi: 10.1242/dev.014068

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A stem-loop structure in the wingless transcript defines a consensus motif for apical RNA transport

Gilberto dos Santos, Andrew J. Simmonds^* and Henry M. Krause

Banting and Best Department of Medical Research, Department of Medical Genetics and Microbiology, University of Toronto, Ontario, M5S 3E1, Canada.

View larger version (38K): [in this window] [in a new window]   Fig. 1. WLE3 mediates apical localization of wg transcripts. (A) Fluorescent deletion constructs of wg 3'UTR RNA were injected into D. melanogaster embryos. Localization efficiency was categorized based on the apical:basal ratio of fluorescent RNA signal as strong (++, ratio 1.5), weak (+, 1.5>ratio1.0) or inactive (-, ratio <1.0). The typical activity of each construct is indicated at the right (* constructs typically inactive but occasionally localized). WLE3 spans nucleotides 518-570, WLE1 57-183, and WLE2 661-776. (B-D) Representative images of embryos injected with fluorescently labelled full-length wg 3'UTR (B), deleted for WLE3 (C) or WLE3 dimer (D). (E-G) Confocal images of stage 11 patched-GAL4::UAS-lacZ embryos containing different regions of the wg 3'UTR fused to lacZ ORF: WLE1-WLE2 (E); WLE1-WLE2-WLE3 (F); WLE3 (G). lacZ reporter transcripts (green) were detected by FISH. Nuclei are red and membranes blue. Scale bars: 10 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 2. Apical wg localization and WLE3 activity is conserved. (A-C) Confocal images of endogenous wg RNA (green) detected by FISH in germ band extended embryos of D. prosaltans (A), D. hydei (B) and Z. tuberculatus (C). Nuclei are red and membranes blue. (D-I) Representative images of D. melanogaster embryos injected with full-length Drosophila wg 3'UTR sequences (D-F), or with chimeric sequences in which the D. melanogaster WLE3 of construct H was replaced by that of D. prosaltans (G), D. hydei (H) or Z. tuberculatus (I). Scale bars: 10 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Predicted WLE3 secondary structure is conserved. (A) Alignment of Drosophila wg 3'UTR sequences with D. melanogaster WLE3 (nucleotides 525-568). Sequences are listed, top to bottom, in order of divergence from D. melanogaster (based on full-length 3'UTR pair-wise comparisons). Invariant residues are shaded black, and bases at variable positions similar to the consensus are shaded grey. Compensatory mutations that preserve base-pairing are shaded green and base-pair disrupting mutations red (*sequences obtained from Drosophila genome projects). (B) Predicted secondary structures for D. melanogaster, D. yakuba, D. prosaltans, D. hydei and Z. tuberculatus WLE3 sequences. Invariant residues are in black text and consensus residues in grey. Non-consensus residues that preserve or disrupt conserved base pairs are green or red, respectively. Non-consensus residues in the loop and central `variable' region are blue. (C) Consensus secondary structure predicted by ALIFOLD. Invariant residues are black, consensus residues grey and positions with conserved base-pairing green.

View larger version (77K): [in this window] [in a new window]   Fig. 4. Importance of sequence and base-pairing at conserved base-pair positions. (A-C) Diagrams of D. melanogaster WLE3 (A) or just the WLE3 distal stem (B,C). Bases targeted by each mutation are boxed in the wild-type diagram, with arrows pointing to mutant sequences shown to either side (mutant numbers/names are indicated at the bottom). The shading indicates activity: white text on black, no activity (all -); white text on dark grey, weak activity (mostly -); black text on light grey, moderate activity (+); black text on white, full activity (++). To the far right of each diagram are representative images of injected embryos. Scale bars: 10 µm. Base transversions are depicted at the immediate left or right of WLE3, and compensatory mutations further right.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Importance of the loop sequence, bulges and the variable region. (A-C) Diagrams of D. melanogaster WLE3 (A,B) or the minimal WLE3 mutant 40.5 (C). Mutant base changes and activities are depicted as in Fig. 4. Representative images of injected embryos are on the right. Scale bars: 10 µm.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Recognition of apical localization elements. Shared features of elements that mediate apical localization in the embryo injection assay. Gene and element names are indicated below each structure. Features shared between the wg, h, ftz, K10, and orb elements are bracketed. The conserved fifth U:A base pair is shaded in light grey. These shared features do not extend to the grk GLS.

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