Analysis of SR proteins that regulate the inclusion of exon 9.

Different SR expression constructs cloned in the pGC vector were co-transfected with the pSPL3-minigenes in HeLa cells to test their effect on NF1 exon 9 splicing. (a) Effect of the overexpression of SR proteins on the wild type minigene. The proportion of transcripts including exon 9 was normalized to the assay of HeLa cells co-transfected with the wild-type pSPL3-minigene and the empty pGC vector (control). The overexpression of SRSF2 and SRSF5 enhanced significantly the inclusion of exon 9. (b) Effect of the overexpression of SRSF5 (SRp40) protein on the mutant minigenes. Comparison of the proportion of transcripts containing exon 9 cotransfecting the minigenes with the empty pCG vector (stripped bars) and with the SRSF5 construct (grey bars). The values have been normalized to the wild type assays. The overexpression of SRSF5 enhanced the inclusion of exon 9 in all the mutant minigenes. (c) Effect of the overexpression of SRSF2 (SC35) protein on the mutant minigenes. Comparison of the proportion of transcripts containing exon 9 in experiments co-transfecting the minigenes with the empty pCG vector (stripped bars) or with SRSF2 constructs (grey bars). The values have been normalized to the wild type assays. Just c.945G>A and c.946C>A mutant minigenes failed to augment the inclusion of exon 9 when SRFS2 was overexpresed. (d) Effect of the increase of SRSF2. pSPL3 mutant minigenes were co-transfected with empty pGC (control, stripped bars) and with increasing amounts of SRSF2 constructs (grey bars). The proportions of transcripts containing exon 9 have been normalized to the controls. SRSF2 rising has no effect on the inclusion of exon 9 from c.945G>A and c.946C>A mutant minigenes whereas there is a gradual increase in the inclusion from c.910C>T mutant. (e) Relative quantification of transcripts including exon 9 upon SRSF2 knockdown. The proportions of transcripts from the minigenes are indicated relative to their respective luciferase controls (stripped bars). Transcripts derived from the WT, c.910C>T and c.943C>T minigenes have a significant reduction in exon 9 inclusion (*** p>0.001) while no effect is observed for c.945G>A and c.946C>A mutant minigenes. In the right panel the qPCR results for the SRSF2 expression levels are shown. SRSF2 levels upon SRSF2 siRNA treatment (grey dotted bars) have been normalized to the SRSF2 levels upon luciferase siRNA treatment (white bars) confirming the effective silencing (*** p<0.001).