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. 2007 Dec 17;28(4):1240–1251. doi: 10.1128/MCB.01509-07

FIG. 1.

FIG. 1.

Hu proteins suppress inclusion of NF1 exon 23a. (a) Diagram depicting the alternative inclusion of exon 23a in the NF1 pre-mRNA. The 63-nucleotide (nt) in-frame exon 23a is specifically excluded in neurons. The arrows indicate the oligonucleotides used to analyze endogenous NF1 splicing by RT-PCR. The sequence of exon 23a and its surrounding introns is shown, with the exon sequence capitalized. AU-rich sequences are underlined. (b) Association of Hu proteins and the NF1 pre-mRNA in CA77 cells. Nuclear extract prepared from CA77 cells was immunoprecipitated with anti-Hu sera. RNA was isolated from either total nuclear extract or immunoprecipitated pellet and analyzed by RT-PCR. Lanes without RT (−) are included as controls. The oligonucleotides used to amplify the NF1 pre-mRNA anneal to sequences in introns upstream and downstream of exon 23a, and the resulting PCR product is 250 nucleotides, while the oligonucleotides used to amplify the GAPDH pre-mRNA anneal to sequences in intron 3 and exon 5, and the resulting PCR product is 585 nucleotides. (c) Correlation of skipping of exon 23a and Hu protein expression. Inclusion of exon 23a in the endogenous NF1 pre-mRNA in HeLa and CA77 cells was detected by RT-PCR. Amplification bands resulting from exon 23a inclusion (267 nucleotides) or exclusion (204 nucleotides) are indicated. Expression of neuron-specific Hu proteins in the two cell lines was detected by Western blot analysis using anti-Hu patient sera. (d) RT-PCR analysis of the NF1 reporter pre-mRNA. HeLa or CA77 cells were cotransfected with the NF1 reporter construct shown in the top panel and increasing amounts (0.5 and 1 μg) of PTB or mHuC. RT-PCR was carried out using total RNA isolated from the transfected cells and the oligonucleotides indicated in the diagram. Amplification products resulting from exon 23a inclusion (309 nucleotides) or exclusion (246 nucleotides) are indicated. The percentage of NF1 exon 23a inclusion is displayed in the bar graphs. The expression of transfected PTB or mHuC in HeLa cells was verified by Western blot analysis. Error bars indicate standard deviations.