Fig. 2.

Role of translin in BDNF mRNA targeting. (A) Conserved translin-binding Y and H elements in human and rat BDNF and mouse Protamine2. (B) Inhibition of the rat and human CDS-GFP RDF values with an siRNA translin mixture (rat ***, P < 0.001; human δδδ, P < 0.001 vs. control), or two siRNA oligos used either alone or in combination (si690, si744), mutated si744 (si744M) had no effect. Inhibition of CDS-GFP dendritic targeting by si690 and si744 was rescued by co-transfection with siRNA-insensitive human translin. (C) Endogenous BDNF mRNA dendritic targeting up-regulation by KCl (***, P < 0.001 Crtl+KCl vs. Crtl), is inhibited by translin siRNA (###, P < 0.001 si-translin+KCl vs. Crtl+KCl), or si690 and si744, but not by scrambled siRNA (NS, not significant). (D) GFP-3′ UTRlong displays an activity-dependent targeting to dendrites in response to 3 h 10 mM KCl that is not affected by si-translin. (E) (Upper) trax antibody immunoprecipitates Translin (arrowhead). Coprecipitation of translin is blocked by omission of Trax antibody or preincubation with its antigen peptide. (Lower) RT-PCR analysis of the immunoprecipitated RNA:protein complexes. BDNF mRNA was detected only in the “offered” sample and the IP pellet containing translin. (F) A radiolabelled segment of the human CDS region containing the Y and H elements (nucleotides 176–211) forms two gel-shift bands after incubation with rat brain extracts. The lower band (arrowhead “Translin”) is “supershifted” with Trax antibody (marked with *). Upper band (small arrow) reflects binding of this probe with an unrelated complex.