A Potential Role for U2AF-SAP 155 Interactions in Recruiting U2 snRNP to the Branch Site

Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3′ splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155’s downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.

U2小核RNA（U2 snRNA）与分支点序列（branchpoint sequence, BPS）之间的碱基配对，对于前体信使RNA（pre-mRNA）的剪接过程至关重要。由于后生动物的BPS序列短小且高度简并，仅靠这一相互作用不足以实现U2小核糖核蛋白（U2 snRNP）的特异性结合。剪接因子U2AF可在最早的剪接体复合物E中结合至3'剪接位点处的嘧啶序列区，且对于剪接体复合物A中U2 snRNP的结合至关重要。本研究证实，在复合物A中，U2 snRNP的SAP 155蛋白可在BPS两侧与前体信使RNA发生紫外交联。SAP 155的下游交联位点紧邻U2AF的结合位点，且二者在蛋白质相互作用实验中可直接发生相互作用。通过紫外交联技术结合对携带重复BPS序列的前体信使RNA的功能分析，我们证实BPS的选择与SAP 155在BPS两侧的紫外交联存在直接相关性。综合来看，我们的实验数据支持如下模型：U2AF首先结合至复合物E中的嘧啶序列区，随后与SAP 155发生相互作用，从而将U2 snRNP招募至BPS处。