Figure 8: AUF-1 facilitates ELAVL4-mediated APP695-specific AS.

Biotinylated RNA probes transcribed from human (A) and mouse (B) APP minigenes were tested for AUF-1 binding. Note, that AUF-1 isoforms p42 and/or p45 bind to all transcripts shown to interact with nELAVLs. (C) SK-N-SH cells were transfected with the DNA3.1 expression vector bearing either no insert (empty) or p42 AUF-1, or with the pENTR/U6 vector carrying a shRNA specific for LacZ or all AUF-1 mRNAs. Inclusion of APP exons 7 and 8 was assayed by RT-qPCR with primers specific for APP770, APP695 and APP751. Total APP cDNA was used for normalization. Bars in graphs correspond to mean ± standard deviation of three independent experiments. (D,E) SK-N-SH cells were co-transfected with the human (D) or the mouse (E) APPE78 minigene along with two expression vectors, one bearing ELAVL4 and the other one carrying no insert (empty) or p42 AUF-1, in ratios depicted. Splicing pathways were determined by RT-PCR, as described in Fig. 5. Quantification of the results was performed by scanning densitometry. Note, that AUF-1 enhances ELAVL4-mediated exclusion of APP exons 7 and 8. (F) Neuro2a cells were transfected with the pSilencer vector bearing either no insert (empty) or a shRNA targeting all Auf-1 transcripts. Inclusion of APP exons 7 and 8 was assayed by RT-qPCR, as described above (**P < 0.01). Note, that in the presence of nELAVLs, reduction of AUF-1 levels results in reduced APP695-specific AS. (G) SK-N-SH cells were co-transfected with two out of three expression plasmids carrying no insert (empty), ELAVL4 or p42 AUF-1 in ratios depicted. Whole cell lysates from these cells were used in a series of pull down assay using streptavidin beads and APP7.4 biotinylated riboprobe. The presence of ELAVLs, AUF-1 and U2AF65 in precipitates was assayed by immunoblotting. Quantification of the results was performed by scanning densitometry and the percentage (mean ± standard deviation) change of U2AF65-binding is shown below each lane. Note that concurrent binding of both proteins in the transcript resulted in a significant reduction of U2AF65 binding. (*P < 0.05, **P < 0.01).