HuD protein specifically recognizes and binds the Msi1 3′UTR sequence. 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 100× 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. Antonia Ratti et al. J Cell Sci 2006;119:1442-1452 © The Company of Biologists Limited 2006