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 studying 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 inclusion of the fibronectin EDI exon into mature mRNA. The T-Ag effect is exon specific, occurs in cis and depends strictly on DNA replication and not on cell transformation. VP16, an activator of transcriptional initiation and elongation, has a similar effect on transcription but the opposite effect on splicing: EDI inclusion is inhibited by 35-fold. VP16 completely reverts the T-Ag effect, but a VP16 mutant with reduced elongation ability provokes only partial reversion. Both T-Ag and VP16 promote conspicuous co-localization of mRNA with nuclear speckles that contain the SR protein SF2/ASF, a positive regulator of EDI inclusion. Therefore, we conclude that co-localization of transcripts and speckles is not sufficient to stimulate EDI inclusion.
Kadener
S
S
Laboratorio de Fisiología y Biología Molecular, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, (C1428EHA) Buenos Aires, Argentina.
Cramer
P
P
Nogués
G
G
Cazalla
D
D
de la Mata
M
M
Fededa
J P
JP
Werbajh
S E
SE
Srebrow
A
A
Kornblihtt
A R
AR
D017398:Alternative Splicing; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000502:physiology*; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D002522:Chlorocebus aethiops; D004261:DNA Replication; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D005091:Exons / Q000235:genetics*; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D017405:Herpes Simplex Virus Protein Vmw65 / Q000502:physiology*; D006801:Humans; D017403:In Situ Hybridization; D008113:Liver Neoplasms / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013539:Simian virus 40; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D004274:DNA, Recombinant; D005353:Fibronectins; D017405:Herpes Simplex Virus Protein Vmw65; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012319:RNA Polymerase II