Fig. 2. Recruitment of Egl and BicD to the HLE. Immunostaining to reveal distribution of Egl (green) and BicD (blue) following injection of h, hΔD or hΔG transcripts (red). Weak apical recruitment of Egl and BicD to hΔG transcripts (which have detectable apical enrichment in 10–20% of embryos) is observed, indicating that a small proportion of the injected transcripts accumulated apically by active transport. Scale bar: 50 µm.

Fig. 1. Mapping the h localization element. (A) Confocal images of representative blastoderm embryos injected with fluorescently labelled transcripts, illustrating examples of wild-type localization (h; ++), no localization (hΔ3′; –) or weak localization (stg-HLE; +). Arrowhead indicates the approximate site of injection. Apical is at the top of this and other figures. (B) Truncated h transcripts map the HLE to between nt 1171 and 1406 (+/– represents very weak apical transcript enrichment in 10–20% of injected embryos). (C) Internal deletions across this region map the HLE to nt 1281–1406. (D) The HLE is sufficient to mediate localization of stg reporter transcripts. Scale bar: 50 µm.

Fig. 4. Characterization of SL1 and SL2a. Site-directed mutagenesis of (A) SL1 and (B) SL2, and representative examples of embryos injected with mutant transcripts as indicated. Mutations are boxed according to the degree of localization they promote. Black, no localization; dark grey, very weak localization in only ∼10% of embryos; light grey, weak localization in all embryos; white, efficient localization. Mutations were tested in the context of the full-length h transcript. (C) Activity of multimers of SL1 (h2xSL1) or SL2a (h2xSL2a, h4xSL2a and h6xSL2a) within the context of the h 3′UTR in embryos fixed 5–8 min after injection. See Supplementary data for the full sequences of the regions used to replace the HLE in these mutants. Scale bar: 50 µm.

Fig. 3. Phylogenetic analysis of the HLE. (A) Localization of h 3′UTR sequences from divergent Drosophila species upon injection into D.melanogaster embryos. Scale bar: 50 µm. (B) Alignment of the RNA sequence of the D.melanogaster HLE with the corresponding region of the h 3′UTR from other Drosophila species. Identical and conserved nucleotides are highlighted in black and grey, respectively. The internal deletions (hΔD-H) of the D.melanogaster HLE are indicated. Dashed lines and thick underscores highlight the sequences forming SL1 and SL2a and the regions mutated in hr21 and hr22 (see Figure 4), respectively. (C) Predicted RNA secondary structures of highly conserved nt 1286–1331 in D.melanogaster h and corresponding sequences in other Drosophila species. Foldings were produced using ConStruct (Lück et al., 1999) using Tinoco settings at 25°C. Nucleotides that differ from D.melanogaster h are highlighted in black. Base-pairing may exist between nucleotides in the illustrated single-stranded regions, although these are not predicted to be evolutionarily conserved.

Fig. 5. Fine structural requirements for SL1 and SL2a. (A) Summary of class III mutations in SL1 (nt 1286–1331). Bases mutated by substitutions or deletions are highlighted in black. Asterisks represent the number of mutations at each position. Mutations that are likely to preserve base pairing that is similar to the wild-type structure (Supplementary figure 2) are boxed. (B) Structure of SL1 in hSLmin1, which supports weak localization. See Supplementary data for the full sequence of the region used to replace the HLE for this mutant. (C) Single base substitutions in SL1 that disrupt localization. 1300^d22C→G does not support localization; 1328^e33A→U and 1316^g15A→U support only weak localization. (D) Summary of class III mutations in SL2a (nt 1380–1402). Mutations are annotated as in (A). In addition, class III mutant g7 contains an insertion of a G after position 1399 (data not shown; Supplementary figure 1). (E) Representative examples of embryos injected with transcripts as indicated. 1328^e33A→U localization is indistinguishable from that of 1316^g15A→U (not shown). Scale bar: 50 µm.

Fig. 6. Real-time analysis of mRNA transport. (A) RNAs are transported intermittently. Tracks of apical–basal motion versus time of representative RNA particles showing predominantly directed (crosses; h 3′UTR) or non-directed motion (triangles; 1328^e33A→U), respectively. A h-SalI3′ particle that lacks a localization signal is also plotted (circles) to indicate the extent of movement of non-localizing particles. Note that movement in the lateral (anterior–posterior) axis is not shown in these plots. Arrows and arrowheads indicate examples of prolonged periods of apical directed and non-directed motion, respectively. (B) Onset of transport of weakly localizing mRNAs is delayed. The y-axis shows the percentage of tracked particles that start directed motion within a specific time window. The data begin 60 s after injection (when imaging began), and is grouped into subsequent bins of 2 min. Imaging was stopped either when no particles were visible basally for >60 s or after 15 min, whichever came first. Total number of particles: wild-type h 3′UTR (wt), 188; h2xSL1, 170; 1328^e33A→U, 184; wt + 1328^e33A→U, 177; 1316^g15A→U, 61; hr11+12, 107. The total RNA concentration was 1 µg/µl for each experiment (500 ng/µl each for wt + 1328^e33A→U). (C) Co-injected h 3′UTR (green; wt) and 1328^e33A→U 3′UTR (red) transcripts being transported in the same particle (arrow) in a living embryo. Transcripts were injected in equimolar amounts. First frame is 4.25 min after injection; subsequent frames are 9.5 s apart. (D) Stills of movies showing overall distribution of h 3′UTR (red; wt) and co-injected 1328^e33A→U 3′UTR (green) transcripts 2.5 min after injection. Total apical accumulation of the wild-type transcript is more efficient than that of the mutant. This can also be seen in (C). Scale bar: 10 µm in (C); 60 µm in (D).