Eperon I C, Makarova O V, Mayeda A, Munroe S H, Cáceres J F, Hayward D G, Krainer A R
Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom.
Mol Cell Biol. 2000 Nov;20(22):8303-18. doi: 10.1128/MCB.20.22.8303-8318.2000.
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 levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. 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 binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream 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.
已知在mRNA前体中识别并区分潜在5'剪接位点（5'SS）的首个组分是U1小核核糖核蛋白颗粒（U1 snRNP）。然而，U1 snRNP与可变5'SS结合的相对水平并不一定决定剪接结果。引人注目的是，必需的SR蛋白剪接因子之一SF2/ASF会导致剪接呈剂量依赖性地转向下游（内含子近端）位点，然而它却同时增加了U1 snRNP在上游和下游位点的结合。我们在此表明，使剪接向上游5'SS转变的hnRNP A1会导致两个位点的U1 snRNP结合减少。尽管如此，U1 snRNP结合的重要性体现在其与下游位点的结合水平和在剪接中的使用之间的比例关系上。使用纯化的组分，hnRNP A1通过与mRNA前体协同且无差别地结合，减少了U1 snRNP与5'SS的结合。hnRNP A1和SF2/ASF中的突变表明，这两种蛋白质对5'SS选择的相反作用与它们对U1 snRNP结合的作用相关。交联实验表明，SF2/ASF和hnRNP A1竞争结合mRNA前体，我们得出结论，这种竞争是它们功能拮抗的基础；SF2/ASF增强了U1 snRNP在所有5'SS上的结合，同时占据率的增加导致剪接转向下游位点，而hnRNP A1干扰U1 snRNP的结合，使得5'SS的占据率较低，并且U1 snRNP对各个位点的亲和力决定了剪接位点。
