FIGURE 8.

Activation of N30 splicing by Fox-3. A, schematic diagrams of NMHC II-B gene, minigene constructs and IDDEs. Native gene shows only a part of the human NMHC II-B gene surrounding exon N30. E5 and E6 are constitutive exons, and N30 and R18 are alternative exons. The IDDE is an intronic region consisting of 201 nucleotides and is located ∼1.5-kb downstream of N30 in the native gene. Rectangles and horizontal lines in the diagrams indicate exons and introns, respectively. Exon size and the IDDE are not drawn to scale. Minigenes include the NMHC II-B genomic DNA fragments indicated by brackets, which are flanked by exons E2 and E3 of the rat preproinsulin gene (PPI). The wild-type (wt) and mutant (ma, mb, and mc) IDDEs are inserted at the indicated location in the minigene. Transcription of the minigene is driven by the Rous sarcoma virus long terminal repeat (RSVLTR). Arrows above E5 and PPIE3 indicate the location of the primers used for RT-PCR. B, dose-dependent activation of N30 splicing by Fox-3. Increasing amounts (0.03, 0.1, 0.3, and 1 μg, indicated by triangle) of the expression constructs encoding Myc-tagged Fox-3-L and -S were co-transfected into SK-N-SH cells with the wild-type minigene. The mRNAs derived from the minigene were analyzed by RT-PCR (upper panel). The upper band includes N30 (+) and the lower band excludes N30 (−). Immunoblots using anti-Myc (lower panel) show relative amounts of the expressed proteins. 0, the empty vector alone; M, molecular size marker. C, UGCAUG-dependent activation of N30 splicing by Fox-3. The minigenes containing wild-type (wt), mutations (ma, mb, and mc) and deletion (−) of the IDDE were co-transfected into SK-N-SH cells with the expression constructs encoding Myc-tagged Fox-3-L and -S. RT-PCR of the minigene mRNAs (upper panel) and immunoblots for the expressed proteins (lower panel) are shown.