Figure 3

αP3A23′G>A compromises binding affinity for a splicing-suppressing hnRNP L and gains binding affinity for a splicing-enhancing hnRNP LL.

(a) Coomassie blue staining of RNA affinity-purified products using SH-SY5Y nuclear extract with biotinylated RNA probes. A ∼70-kDa protein (arrow) is detected only with the wild-type (wt) probe but not with the scrambled (scr) probe. (b) Immunoblots (IB) probed with candidate splicing trans-factors. The wild-type exon P3A (wt) binds to hnRNP L (L) and the αP3A23′G>A (mut) disrupts its binding. The mutation gains de novo binding to hnRNP LL (LL). NE indicates 10% of nuclear extract used. (c) Deletion mutagenesis to map the binding sequences of hnRNPs L and LL in exon P3A. Underlines indicate putative binding motifs for hnRNP LL (red lines) and hnRNP L (thin and thick blue lines for weak and strong motifs, respectively)²⁷. Note that the G-to-A mutation de novo generates a binding motif of CACC for hnRNPs L and LL. Immunoblots of RNA affinity-purified products are detected with antibodies against hnRNPs L and LL. (d) Mock-, hnRNP L- and hnRNP LL-depleted SH-SY5Y nuclear extracts were affinity-purified with RNA probes and resolved by immunoblotting. (e) RT-PCR of wild-type (wt) and mutant (mut) pSPL3 minigenes in SH-SY5Y cells treated with siRNA against control (siCt), hnRNP L (siL) and hnRNP LL (siLL). Immunoblotting shows down regulation of hnRNPs L and LL. (f) Schematic presentation of a reporter minigene (pSPL3-MS2) and trans-acting effectors. HnRNPs L and LL (ovals) are fused to the artificial MS2 coat protein (inverted U shape). MS2 coat protein-binding hairpin RNA is substituted for the splicing regulatory site of exon P3A to directly tether MS2 coat protein-fused hnRNPs L and LL. RT-PCR of pSPL3-MS2 minigenes in SH-SY5Y cells that are co-transfected with the indicated effectors. Immunoblotting shows expression of effectors in the nuclear lysate of SH-SY5Y cells. The MS2-L construct has 16 additional codons arising from the multiple cloning site compared to MS2-LL. Bars and lines represent mean and SD, respectively, of three independent experiments for panels (e) and (f).