An alternative, but not exclusive, model suggests that transcription might control splicing via the regulation of pol II elongation rate or processivity. Low pol II elongation rate or internal pauses for elongation would favor the inclusion of alternative exons governed by an exon skipping mechanism, whereas a highly elongating pol II, or the absence of internal pauses, would favor exclusion of these kinds of exons. The mechanism by which the elongation rate would affect EDI splicing is a consequence of EDI pre-mRNA sequence. EDI exon skipping occurs because the 3'-splice site of the upstream intron is suboptimal compared with the 3'-splice site of the downstream intron. If the polymerase pauses anywhere between these two sites, only elimination of the upstream intron can take place. Once the pause is passed or the polymerase proceeds, there is no option for the splicing machinery but to eliminate the downstream intron, which leads to exon inclusion. A highly processive elongating pol II, or the absence of internal pauses, would favor the simultaneous presentation of both introns to the splicing machinery, a situation in which the stronger 3'-splice site of the downstream intron outcompetes the weaker 3'-splice site of the upstream intron, resulting in exon skipping. Figure 3
shows how when a weak 3'-splice site is followed by a strong one, as in many alternative splicing examples, pol II elongation rates affect the relative amounts of splicing isoforms. On the contrary, when two consecutive strong 3'-splice sites occur, as in constitutive splicing, pol II elongation rates are irrelevant.
A kinetic role for transcription on splicing was originally suggested by Eperon et al. (1988), who found that the rate of RNA synthesis may affect its secondary structure, which may, in turn, affect splicing. A similar mechanism involving a kinetic link between transcription and splicing was suggested from experiments in which RNA pol II pause sites affect alternative splicing by delaying the transcription of an essential splicing inhibitory element (DRE) required for regulation of tropomyosin exon 3 (Roberts et al. 1998).
The elongation factor P-TEFb converts the polymerase from a nonprocessive to a processive form, which is consistent with the fact that inhibitors of this kinase such as DRB (dichlororibofuranosylbenzimidazole) or flavopiridol inhibit pol II elongation (Price 2000; Ni et al. 2004). Cells transfected with EDI splicing reporters and treated with DRB displayed a threefold increase in EDI inclusion into mature mRNA compared with untreated cells (Nogués et al. 2002).
Changes in chromatin structure provoked by histone acetylation also affect splicing. In fact, trichostatin A, a potent inhibitor of histone deacetylation, inhibits EDI inclusion (Nogués et al. 2002). This supports the hypothesis that acetylation of the core histones would facilitate the passage of the transcribing polymerase, which is, in turn, consistent with the proposal of chromatin opening mediated by DNA tracking by a transcribing pol II complex piggybacking a histone acetyltransferase activity (Travers 1999).
The above mentioned weakness of the 3'-splice of the upstream intron of EDI is caused by its suboptimal polypyrimidine tract. By mutating EDI’s polypyrimidine tract and therefore generating pre-mRNAs with increasing degrees of exon recognition, it was shown that responsiveness of exon skipping to elongation is inversely proportional to the 3'-splice site strength, which means that the better the alternative exon is recognized by the splicing machinery, the less its degree of inclusion is affected by transcriptional elongation (Nogués et al. 2003).
Answers to all your Biology Questions
