Figure 6: nELAVL-binding to sequences located both upstream and downstream of exon 7 is required for nELAVL-mediated APP exon 7 exclusion. | Scientific Reports

Figure 6: nELAVL-binding to sequences located both upstream and downstream of exon 7 is required for nELAVL-mediated APP exon 7 exclusion.

From: Neuronal ELAVL proteins utilize AUF-1 as a co-partner to induce neuron-specific alternative splicing of APP

Figure 6

(A,B) Schematic representation of human (A) and mouse (B) APP genomic regions used for the generation of APPE7 and AppE7 minigenes, respectively. The gray box corresponds to exon 7, bold lines to its flanking intronic sequences, whose length is indicated and triangles to segments encoding for U-rich sequences in the pre-mRNA. (C,D) Human SK-N-SH cells were co-transfected with either APPE7 (C) or AppE7 (D) minigenes and the pCAGGS expression vector bearing ELAVL4. Splicing pathways were determined by RT-PCR using primers specific for the artificial exons of the minigene vector. Amplification bands of transcripts lacking exon 7 are shown. Quantification of the results was performed by scanning densitometry. Bars in graphs depict the percentage of exon 7 skipping (mean ± standard deviation). Note, that ELAVL4 promoted exon 7 exclusion only from transcripts containing U-rich sequences both upstream and downstream of this exon (E,F) Biotinylated RNA probes transcribed from the indicated human (E) and mouse (F) APPE7 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 both flanking intronic regions of APP exon 7.

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