Transcriptional activators differ in their abilities to control alternative splicing.
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.
Nogues
Guadalupe
G
Laboratorio de Fisiologia y Biologia Molecular, Departamento de Fisiologia, Biologia Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón II, C1428EHA Buenos Aires, Argentina.
Kadener
Sebastian
S
Cramer
Paula
P
Bentley
David
D
Kornblihtt
Alberto R
AR
D017398:Alternative Splicing; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D004742:Enhancer Elements, Genetic; D005091:Exons; D005353:Fibronectins / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D012319:RNA Polymerase II / Q000235:genetics; D018741:Replication Origin; D013539:Simian virus 40 / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D005353:Fibronectins; D015534:Trans-Activators; D012319:RNA Polymerase II