Selection of alternative 5 ' splice sites: Role of U1 snRNP and models forthe antagonistic effects of SF2/ASF and hnRNP A1

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative lev els of U1 snRNP binding to alternative 5'SSs do not necessarily determine t he splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-s plicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream a nd downstream sites simultaneously. We show here that hnRNP A1, which shift s splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at bot h sites. Nonetheless, the importance of U1 snRNP binding is shown by propor tionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP bi nding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRN A. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP bindi ng. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bin d pre-mRNA, and we conclude that this competition is the basis of their fun ctional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the ris e in simultaneous occupancy causing a shift in splicing towards the downstr eam site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.