Regulation of alternative 3' splice site selection by constitutive splicing factors.
We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005091:Exons; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D038941:Polypyrimidine Tract-Binding Protein; D012326:RNA Splicing; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D000072668:Splicing Factor U2AF; D013347:Subcellular Fractions / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; D014335:Tropomyosin; C000607494:U2AF2 protein, human; D038941:Polypyrimidine Tract-Binding Protein; D000068103:Serine-Arginine Splicing Factors