Fig. 5. HuD protein specifically recognizes and binds the Msi1 3'UTR sequence. (A) Schematic representation of the mouse Msi1 3'UTR (399 bp). ARE⁺ (230 bp) and ARE^- (148 bp) deletion segments were obtained by PCR amplification with the indicated primer pairs (see Materials and Methods). (B) Left panel, polyacrylamide gel of UV-crosslinking assays on neurosphere extracts with the ARE⁺ and ARE^- deletion segments. The binding activity was completely abolished in the absence of the ARE element. Msi1 full-length 3'UTR was used as a positive control (middle lane). Right panel, UV crosslinking assay of the recombinant HuD protein with the Msi1 3'UTR riboprobe compared with Gap43 as a control. The 42 kDa complex (arrowhead) can be detected. HuD specifically bound the deletion fragment ARE⁺, but not the ARE^- sequence. (C) Left panel, competition experiments with the recombinant HuD protein were performed with the addition of a 100x molar excess of cold ARE⁺ and ARE^- riboprobes. The Msi1-HuD complex is shown in the first lane before the addition of the cold competitors. Right panel, an excess of cold full-length Msi1 3'UTR completely inhibited the formation of the mRNP complex. The antisense Msi1 sequence (As) was not able to specifically bind HuD protein. (D) Dose-dependent binding-activity of the HuD protein (0.1, 0.2, 0.4, 1 µg) to the Msi1 3'UTR sequence.