Figure 1. Antisense to ß-Actin Zipcodes Inhibits Signaling of ß-Actin mRNA Localization to Growth Cones by NT-3

(A) Schematic illustration showing the position of the 54 nt and 43 nt ß-actin mRNA localization sequences (zipcodes) within the 3'UTR (Kislauskis et al., 1994). Two antisense oligonucleotides were designed to each of the zipcodes and used in combination. Coding region (black), 3'UTR (green), zipcodes (red).

(B–E) In situ hybridization to detect ß-actin mRNA (red) in neurons that were starved or stimulated with NT-3 in the presence of antisense or reverse antisense oligonucleotides, as a control. Starved neurons did not show hybridization signal for ß-actin mRNA in growth cones (B and D; arrows). Addition of NT-3 (25 ng/ml) to the starvation medium for 30 min in the presence of the antisense oligonucleotides failed to show ß-mRNA localization in growth cones (C, arrow). In contrast, addition of NT-3 in the presence of the control oligonucleotides demonstrated strong signaling of ß-mRNA localization to distal neurites and growth cones (E, arrows).

(F) The percentage of cells which exhibited ß-actin mRNA within growth cones.

(G) The measurement of the fluorescence intensity (mean pixel intensity) of ß-actin mRNA signal within growth cones. Both methods demonstrated a statistically significant increase in ß-actin mRNA localization following NT-3 treatment, in the presence of the control oligonucleotides (p < 0.01, Student's t test).

(H) Northern Blot analysis of ß-actin mRNA levels in normal culture conditions with N2 supplements, following starvation in MEM, 60 min in NT-3, with or without antisense treatment.