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. 2013 May 18;41(14):6793–6807. doi: 10.1093/nar/gkt421

Figure 4.

Figure 4.

Mutagenesis validates predicted YCAY clusters. Mutagenesis analyses of Nova-binding YCAY clusters were previously performed in 293T or N2A cells for three splicing reporters. In each case, coordinates and schematic representation of the exon and intron structure, sequence conservation, CLIP tags and predicted YCAY clusters, as well as mutations introduced in the reporters are shown in the left panel. YCAY clusters predicted by our previous analysis (22) is indicated by a solid box in the YCAY track. The splicing of each reporter with WT or mutant YCAY clusters, in combination with transfection of Nova plasmids in N2A and/or 293T cells, was quantified by RT-PCR. Exon inclusion level of each reporter (y-axis) is correlated with the WT or mutant YCAY cluster score (x-axis), as shown on the right. The squared Pearson correlation coefficient is indicated. (A) Gabrg2 exon 9 (10). The minigene consists of sequences between exons 8 and 10, as shaded in gray in the schematic representation of the gene structure. Mutant minigenes were generated by point mutations in the different sets of YCAY elements (YCAY→YAAY), as indicated by the red boxes with a cross. The analysis was performed in both N2A cells and 293T cells. (B) Nova1 exon4 (9). The minigene constructs consist of Nova1 exon 4 and flanking intronic sequences inserted into the human β-globin gene backbone. Mutant minigenes were generated by truncation of intronic sequences of different sizes covering the predicted YCAY clusters, together with point mutations in the YCAY elements (YCAY→YAAY), as indicated by the red boxes with a cross. The analysis was performed in both N2A cells and 293T cells. (C) Dab1 exons 7b and c (41). The minigene constructs consist of exons 7b and c and flanking intronic sequences inserted into the human β-globin gene backbone. Mutant minigenes were generated by point mutations in different sets of YCAY elements (YCAY→YAAY), as indicated by the red boxes with cross. The analysis was performed in 293T cells. Inclusion of both exons 7b and c is shown.