Figure 5: Identification of a U-rich element important for nELAVL-mediated APP exon 8 skipping.

(A,B) Schematic diagrams depicting the regions of human (A) and mouse (B) APP locus used for the generation of APPE8 and AppE8 minigenes, respectively. The gray box represents exon 8, bold lines its flanking intronic sequences, whose length is depicted, and triangles segments encoding for U-rich regions in the pre-mRNA. The 47 nt upstream sequence of wild-type and mutant APPE8.4 and AppE8.5 is also shown. (C,D) Human SK-N-SH cells were co-transfected with human (C) or mouse APPE8 (D) minigenes and the pCAGGS expression vector bearing no insert (empty) or ELAVL4. Splicing pathways were determined by RT-PCR using primers specific for the artificial exons of the minigene vector. Amplification bands resulting from exon 8 skipping are indicated. Quantification of the results was performed by scanning densitometry and the percentage of exon 8 exclusion (mean ± standard deviation) is displayed in graphs (**P < 0.01, ***P < 0.001). Note that ELAVL4 efficiently promoted exclusion of exon 8 from all wild-type artificial transcripts, but this effect was compromised in the case of mutant ones. (E,F) Biotinylated RNA segments transcribed from wild-type and mutant APPE8.4 (E) and AppE8.5 (F) minigenes were tested for nELAVL-binding after incubation with lysates of Neuro-2a cells and pull-down using streptavidin beads. The presence of nELAVLs was assayed by immunoblotting. Note that nELAVLs strongly associated with the wild-type transcripts, but this association was weaker when the mutant transcripts were used.