Antagonistic effects of T-Ag and VP16 reveal a role for RNA pol II elongation on alternative splicing

Here we investigate the promoter control of alternative splicing by studyin g two transcriptional activators on templates under replicating conditions. SV40 large T-antigen (T-Ag) activates template replication only 2-fold but transcription 25-fold. T-Ag-mediated replication, reported to inhibit RNA polymerase II elongation, provokes a 10- to 30-fold increase in the inclusi on of the fibronectin EDI exon into mature mRNA. The T-Ag effect is exon sp ecific, occurs in cis and depends strictly on DNA replication and not on ce ll transformation. VP16, an activator of transcriptional initiation and elo ngation, has a similar effect on transcription but the opposite effect on s plicing: EDI inclusion is inhibited by 35-fold. VP16 completely reverts the T-Ag effect, but a VP16 mutant with reduced elongation ability provokes on ly partial reversion. Both T-Ag and VP16 promote conspicuous co-localizatio n of mRNA with nuclear speckles that contain the SR protein SF2/ASF, a posi tive regulator of EDI inclusion. Therefore, we conclude that co-localizatio n of transcripts and speckles is not sufficient to stimulate EDI inclusion.