2082 Nucleic Acids Research, 2005, Vol. 33, No. 7 Figure 3. Subcellular distribution of the Fxh and A2BP1 isoforms. The myc-tagged proteins indicated were exogenously expressed in HeLa cells (A) and Y79 cells (B). Immunofluorescence confocal images co-stained with an antibody to the myc-epitope (red) as well as those to NMHC-B (green) and DAPI (blue, for nuclei) are shown. The two bottom images in each panel in (B) are overlays of red with blue (left) or green (right). exogenously expressed isoforms of Fxh and A2BP1 show a similar subcellular distribution as in HeLa cells (Figure 3B). F011 localizes predominantly to nuclei, A016 to both nuclei and cytoplasm and A030 predominantly to cytoplasm. Specific interaction of Fxh and A2BP1 with IDDE via a hexanucleotide UGCAUG Fxh and A2BP1 share an identical RRM and A2BP1 has been reported to bind speciﬁcally to the pentanucleotide GCAUG through this RRM (21). We have previously reported that the IDDE of the NMHC-B transcript, which is indispensable for the regulation of neural cell-speciﬁc cassette type exon N30 splicing, has two copies of GCAUG (14). Therefore, we investigated whether Fxh and/or A2BP1 bound to the IDDE. Electrophoretic mobility shift assays (EMSAs) were carried out using labeled IDDE and in vitro transcribed and translated Fxh and A2BP1, which include a myc-epitope. Since all Fxh and A2BP1 isoforms, except F402 and A704, contain the identical RRM, representative isoforms were ana- lyzed. The expression of F011 in reticulocyte lysate causes the formation of a RNA–protein complex whose migration shift distinguishes it from those of the control reticulocyte lysate (C in Figure 4B, lanes 3 and 10). The unlabeled wild-type IDDE competes with the probe efﬁciently for the formation of the speciﬁc complex C (Figure 4B, lane 4). On the other hand, the mutant mc (Figure 4A), which has a mutation in both copies of UGCAUG, does not (Figure 4B, lane 7). The mutant ma, which has a mutation in the hexanucleotide at the 50 side, shows less efﬁcient competition, compared with mb, which has a mutation in the 30 side of the hexanucleotide (Figure 4B, lanes 5 and 6), indicating that the nucleotides at the 50 side are more important than those at the 30 side. The presence of an anti-myc antibody, but not a non-speciﬁc antibody, inhibits the formation of the speciﬁc complex (Figure 4B, lane 8). Syn- thesis of the full-length F011 protein using a reticulocyte lysate and speciﬁcity of the myc antibody for the expres- sed protein are veriﬁed by immunoblot analysis (Figure 4B, lanes 11 and 12). A016 and A030 show essentially identical results to those with F011 (data not shown). These results indicate that Fxh and A2BP1 can bind to the IDDE and that the hexanucleotide UGCAUG is required for their binding. Fxh and A2BP1 enhance N30 inclusion in a UGCAUG-dependent manner To study whether Fxh and A2BP1 regulate neural cell-speciﬁc splicing via binding to UGCAUG, the Fxh and A2BP1 expres- sion constructs were co-transfected into retinoblastoma Y79 cells with a number of the reporter minigene constructs, which include the wild-type or a mutant version of UGCAUG in the IDDE (Figure 4A). Minigenes H and J consist of the exons E5, N30 and E6 and their ﬂanking introns with some deletions in the introns. The IDDE with or without mutations in the