Differential ASF/SF2 activity in extracts from normal WI38 and transformed WI38VA13 cells.
The normal human fibroblast cell line WI38 and a transformed derivative, WI38VA13, differentially splice fibronectin pre-mRNA in vivo. As a first step to understand the molecular basis for this regulation of splicing, we examined the ability of WI38 and WI38VA13 nuclear extracts to splice model adenovirus and globin pre-mRNAs. Adenovirus RNA splicing was detected in WI38VA13 but not in WI38 extracts. Likewise, when supplemented with a HeLa post-nuclear supernatant (S100), human beta-globin RNA splicing was detected in WI38VA13 but not in WI38 extracts. The splicing defect in WI38 extracts was associated with a reduced ability to form splicing complexes and with a corresponding decrease in the interaction of U2 small nuclear ribonucleoprotein (snRNP) with the branchsite. These defects did not correlate with a decrease in 65 kD U2AF binding since equivalent U2AF level and activity were detected in WI38 and WI38VA13 extracts. Rather, WI38 extracts displayed reduced ASF/SF2 activity and contained a low level of 30 and 40 kD SR phosphoproteins. Moreover, addition of purified ASF/SF2 dramatically increased splicing complex formation in WI38 extracts. These results raise the possibility that variations in the level and activity of ASF/SF2 and other SR proteins play a role in the regulation of fibronectin splicing.
Chabot
B
B
Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Frappier
D
D
La Branche
H
H
D017398:Alternative Splicing; D002461:Cell Line, Transformed; D005353:Fibronectins / Q000235:genetics*; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D016601:RNA-Binding Proteins; D006163:Ribonuclease T1; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D005353:Fibronectins; D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D017413:Ribonucleoprotein, U2 Small Nuclear; D000068103:Serine-Arginine Splicing Factors; D005914:Globins; D006163:Ribonuclease T1