Fig 2: Differential expression of RBFOX-target exons in rat brain cortex (COR) and suprachiasmatic nucleus (SCN), and differential activity of RBFOX2 isoforms. (A) Representative agarose gel electrophoresis images of RT-PCR analysis using primers directed at transcript regions spanning exons that are documented as either included (Grin1 exon 4, Enah exon 12, or excluded (Cacna1c exons 7 & 29) by RBFOX protein activity. Exons are numbered according to currently annotated rat gene transcripts (see text). Numbers on right indicate amplicon sizes in bp. (B) Schematic representation of gene exon structure. Arrows indicate position of proximal (within 500 nt) UGCAUG (consensus RBFOX-binding) sequences. (C) Representative agarose gel electrophoresis images of RT-PCR analysis of PC12 cell RNA showing the effect of RBFOX2 (RBF2) over-expression (vs. emply vector, pc3.1) on exon usage in target genes. Note the marked reduction in Cacna1c exon 7 inclusion in the RBF2 lane, but no effect on Cacna1c exon 7. (D) Comparative analysis of non-variant RBFOX2 (RBF2) and -12–40 RBFOX2 variant (-12–40) over-expression on Cacna1c exon 7 exclusion in PC12 cells. Upper: Representative agarose gel electrophoresis images of RT-PCR analysis. Note the Rbfox2 primers are specific for exogenous Rbfox2 transcripts. Lower: Quantitative comparison of multiple experiments showing a significant reduction of exon 7 inclusion with RBF2 (*p < 0.05 [p < 0.000, 2-tailed] Independent Student’s t test) but no significant effect of the -12–40 variant (p > 0.05 [p = 0.743, 2-tailed] Independent Student’s t test. Levene’s test for equality of variance: RBF2: F = 6.179, p = 0.047; -12–40: F = 7.762, p = 0.032. Values are Mean ± SEM; n = 4 replicate transfections in each group. Note that gel images have been cropped for clarity; full-length gel images for each amplicon are shown in Fig. S4.

Fig 3: Rbfox2 is highly expressed in rat brain, and multiple isoforms are derived from a multi-exon transcript that includes a ‘MEKK’ ATG start. (A) Representative agarose gel electrophoresis images of RT-PCR analysis using forward primers directed against two different Rbfox2 start sites represented as ‘MEKK’ and ‘MAEG’. Left: abundance of ‘MEKK’ isoforms compared with ‘MAEG’ isoforms in rat cerebral cortex (COR). Centre: similar abundance of ‘MEKK’ isoforms in cortex and suprachiasmatic nucleus (SCN). Right: abundance of ‘MEKK’ isoforms in PC12 cells. PCR product size is estimated against either Hyperladder I (left and centre) or Promega 1 kb ladder (right). (B) Schematic representation of cloned rat brain Rbfox2 cDNA structure showing (predicted) alternative 5' exons and approximate coding positions of the protein domains encoded by ‘MEKK’ isoforms (N-terminal, NTD; RNA recognition motif domain, RRM; C-terminal, CTD). The ‘MAEG’ ‘exon’ (E) is shown dashed to indicate that there may be more than 1 exon in this alternative 5' start. Exons and introns are not to scale. (C) Schematic representation of rat brain Rbfox2 cDNA structure showing sites of exon variation (dashed boxes), and alternative exon insertions (bold italic numbers) in cloned rat brain Rbfox2 cDNAs. Arrows indicate the location of proximal intronic (within 500 nt) UGCAUG (consensus RBFOX-binding) sequences.