Alternative 3' splice-site selection using HeLa cell nuclear extracts prepared with high-ionic buffers.
To investigate the role of cellular trans-acting factors in alternative 3' splice-site selection, a series of HeLa cell nuclear extracts were generated with salt washes ranging from 0.4 to 0.8 M salt. These extracts were tested with human beta-globin pre-mRNAs containing tandem 5' or 3' splice-site duplications as the substrates. High-salt (0.6 M and higher)-based buffers generated nuclear extracts that differentially processed pre-mRNAs containing competing 3' splice sites. High-salt extracts increased the usage of the distal 3' splice site, whereas no shift in 5' splice-site usage could be detected. Western analysis suggested that this shift in alternative 3' splice-site selection was not due to changes in the U2 snRNP auxiliary factor or polypyrimidine tract-binding protein levels.
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
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Zerivitz
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JP
Kreivi
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Akusjärvi
D017398:Alternative Splicing; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005914:Globins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins; D038941:Polypyrimidine Tract-Binding Protein; D012322:RNA Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012342:RNA, Small Nuclear / Q000235:genetics; D016601:RNA-Binding Proteins / Q000502:physiology; D012261:Ribonucleoproteins / Q000502:physiology; D000072668:Splicing Factor U2AF; D015533:Transcriptional Activation
D009687:Nuclear Proteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D038941:Polypyrimidine Tract-Binding Protein; D005914:Globins