Nucleic Acids Research, 2005, Vol. 33, No. 7 2083 Figure 4. (A) Schematic diagrams of minigene constructs and IDDE. Rectangles and solid lines in the diagrams indicate exons and introns, respectively. The broken lines indicate the deleted intron regions. The NMHC-B gene is flanked by the preproinsulin (PPI) exons (E) 2 and 3 in the minigenes. Exon and intron sizes are not in scale. The wild-type (wt) and mutant (ma, mb and mc) IDDEs were inserted at the indicated location of minigenes J and H. Minigene G contains the 13 kb fragment of the native gene. (B) UGCAUG-dependent interaction of Fxh with IDDE. EMSA was carried out using labeled wild-type IDDE as a probe and myc-tagged F011 protein (Prot. +), which were expressed in reticulocyte lysate. The specific probe–protein complex (C) and the free probe (F) are indicated. The unlabeled wild-type (wt) and mutant (ma, mb and mc) IDDEs shown in (A) were used as competitors (Comp.) at a concentration of 30-fold molar excess relative to the probe. Ab, antibody; NS, non-specific; R, reticulocyte lysate alone; and , no addition. Synthesis of the full-length protein in a reticulocyte lysate is verified by immunoblot analysis using an anti-myc antibody following SDS–PAGE. (C) UGCAUG-dependent activation of N30 splicing by Fxh and A2BP1. The minigenes indicated were co-transfected with the expression constructs for the indicated proteins in Y79 cells. The mRNAs derived from the minigenes were analyzed by RT–PCR using primers P7 and P8 indicated in (A). The 173 bp products include N30 (N30, +) and the 143 bp products exclude N30 (N30, ). Immunoblots using an anti-myc antibody show relative amounts of the expressed proteins. hexanucleotide is included or excluded between N30 and E6. As described above, each isoform of Fxh and A2BP1 enters the nucleus to a different extent. To see the effects of different proteins on N30 splicing itself, independent of their differen- tial properties in nuclear localization, an exogenous NLS was added to the expressed proteins. Since Fxh and A2BP1 pro- teins contain the identical RRM, and F011, A016 and A030 all show the same UGCAUG-dependent binding to the IDDE in vitro, F011 and A030 were used for these experiments. Host cells Y79 at the proliferating stage exclude the N30 exon in 90% of the endogenous NMHC-B mRNAs (e.g. see Figure 6A, lane 1). As shown in Figure 4C, the mRNAs derived from minigene J exclude N30 without exogenous expression of Fxh or A2BP1 in Y79 cells, similar to the endogenous NMHC-B mRNAs (Figure 4C, upper panel, lanes 1–5). However, in the presence of exogenous expression of F011 and A030, the N30 inclusion is increased (Figure 4C, upper panel, lanes 7 and 12). The N30 inclusion is absolutely dependent on the presence of the IDDE (Figure 4C, upper panel, lanes 6 and 11). Moreover, mutation