Abstract
Promoter and enhancer elements can influence alternative splicing, but the basis for this phenomenon is not well understood. Here we investigated how different transcriptional activators affect the decision between inclusion and exclusion (skipping) of the fibronectin EDI exon. A mutant of the acidic VP16 activation domain called SW6 that preferentially inhibits polymerase II (pol II) elongation caused a reduction in EDI exon skipping. Exon skipping was fully restored in the presence of the SW6 mutant by either the SV40 enhancer in cis or the human immunodeficiency virus (HIV) Tat in trans, both of which specifically stimulate pol II elongation. HIV Tat also cooperated with the Sp1 and CTF activation domains to enhance transcript elongation and EDI skipping. The extent of exon skipping correlated with the efficiency with which pol II transcripts reach the 3' end of the gene but not with the overall fold increase in transcript levels caused by different activators. The ability of activators to enhance elongation by RNA polymerase II therefore correlates with their ability to enhance exon skipping. Consistent with this observation, the elongation inhibitor dichlororibofuranosylbenzimidazole (DRB) enhanced EDI inclusion. Conversely, the histone deacetylase inhibitor trichostatin A that is thought to stimulate elongation caused a modest inhibition of EDI inclusion. Together our results support a kinetic coupling model in which the rate of transcript elongation determines the outcome of two competing splicing reactions that occur co-transcriptionally. Rapid, highly processive transcription favors EDI exon skipping, whereas slower, less processive transcription favors inclusion.
Highlights
The model is supported by the following evidence. (a) Promoters eliciting higher EDI inclusion levels determine lower polymerase II (pol II) processivities than promoters eliciting lower EDI inclusion, as revealed by the measurements of pol II densities along the minigene templates by chromatin immunoprecipitation [14]. (b) A few rounds of minigene replication, which causes a decrease in pol II processivity presumably by chromatin compaction, provokes higher inclusion of the alternative EDI exon [15]. (c) The
VP16, SV40 Enhancer, T-antigen-dependent Replication, and Alternative Splicing—When ␣-globin/FN minigenes containing the SV40 enhancer and origin of replication are transfected into COS-1 cells, which constitutively express SV40 T-antigen, RNA pol II elongation is controlled by both viral elements
In a previous report [15] we showed that transcriptional activation by Gal4VP16 targeted to pSVEDA/Gal5-human immunodeficiency virus (HIV)-2 minigene (Fig. 1A) inhibits EDI inclusion in COS-1 cells
Summary
Plasmid Constructs—pSVEDA/Gal5-HIV-2 has been described previously [13] This plasmid contains the ␣-globin/FN minigene reporter for alternative splicing of the EDI exon under the control of the HIV-2 promoter fused to five copies of the target site for the DNA binding domain of Saccharomyces cerevisiae Gal. This plasmid contains the ␣-globin/FN minigene reporter for alternative splicing of the EDI exon under the control of the HIV-2 promoter fused to five copies of the target site for the DNA binding domain of Saccharomyces cerevisiae Gal4 This plasmid generates transcripts with the Tar sequence at their 5Ј ends, which is a binding site for HIV-1 or HIV-2 Tat. The construct contains a SV40 enhancer/origin (e/o) located approximately at Ϫ600 bp with respect to the transcriptional start site. RNase Protection Assay (RPA)—The design of distal and proximal riboprobes and RPA conditions were described previously [15]
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