Alternative splicing (AS) is a major contributor to proteome diversity in metazoans. AS of the fibronectin extra domain I (EDI) exon is controlled by RNA polymerase II (pol II) elongation and by the binding of the splicing factor SF2/ASF to an exonic splicing enhancer (ESE) present in EDI. Faster elongation rates elicit higher exon skipping according to a model where skipping occurs because the 3′ splice site (ss) of the upstream intron is suboptimal compared with the 3′ss of the downstream intron. We tested the model in cis, by strengthening the suboptimal 3′ss, and in trans, by looking at AS when transcription is performed by a "slow" pol II mutant. We found that the better the exon is recognized by the splicing machinery, the less its splicing is affected by pol II elongation (Nogués et al., J. Biol. Chem. 278:52166–52171, 2003) and that transcription by a "slow" polymerase not only inhibits EDI skipping but also affects alternative splicing of other genes such as adenovirus E1a and Drosophila's Ultrabithorax (de la Mata et al., Mol. Cell 12:525–532, 2003).
We prepared minigenes with two EDI regions arranged in tandem under a single promoter. We found that the proximal AS event influences the distal one: disruption of the ESE at the proximal EDI not only prevents its own inclusion but favors skipping of a wild-type distal EDI exon. The effect is polar in nature because disruption of the distal EDI ESE does not affect skipping of the proximal one, which suggests a strong link with transcription.
The human transcriptional coactivator CA150 was reported to decrease transcriptional elongation by interacting with pol II carboxy-terminal domain. Consistent with our model, overexpression of CA150 provokes a fourfold increase in EDI inclusion. Paradoxically, reduction in endogenous levels of CA150 by RNA interference also increases EDI inclusion, indicating dual roles for this coactivator.