NeuN/Rbfox3 Nuclear and Cytoplasmic Isoforms Differentially Regulate Alternative Splicing and Nonsense-Mediated Decay of Rbfox2
Figure 5
Rbfox3 variants all function to regulate alternative splicing, irrespective of steady-state sub-cellular localization.
N2A or 293T cells were transiently transfected with Rbfox3 variants 1, 2 or 3 harboring N-terminal flag-tags, or pcDNA3 vector control. A. Alternative splicing of endogenous mRNAs encoding Rbfox2, c-src and CaV1.2 (alternative exons 9* and 33) was assayed by RT-PCR. Rbfox2 +e6* represents splicing from exon 5 to a cryptic exon downstream of exon 6. B. Western with anti-flag was used to confirm Rbfox3 overexpression, and anti-actin was used as a loading control. C. Quantification of the results shown in A and 2 additional experiments (i.e. n = 3); results are displayed as average +/− standard deviation. All Rbfox3 variants promoted skipping of Rbfox2 exon 6, but no change was seen in the splicing of c-src exon N or CaV1.2 exons 9* or 33 upon Rbfox3 overexpression.