PATHWAYS FOR SELECTION OF 5' SPLICE SITES BY U1 SNRNPS AND SF2 ASF/

We have used protection against ribonuclease H to investigate the mech anisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial bind ing of U1 snRNPs to alternative consensus splice sites was indiscrimin ate, and on a high proportion of pre-mRNA molecules both sites were oc cupied simultaneously. When the sites were close, this inhibited splic ing. We propose that double occupancy leads to the use of the downstre am site for splicing and that this is the cause of the proximity effec t seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity o f U1 snRNPs for the downstream site. This prediction was tested both b y cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstre am 5' splice site use by splicing factor SF2/ASF appears to be mediate d by an increase in the strength of U1 snRNP binding to all sites indi scriminately.