Cáceres J F, Krainer A R
Cold Spring Harbor Laboratory, NY 11724-2208.
EMBO J. 1993 Dec;12(12):4715-26. doi: 10.1002/j.1460-2075.1993.tb06160.x.
Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.
人类前体mRNA剪接因子SF2/ASF具有体外一般剪接所需的活性，并通过对抗hnRNP A1以浓度依赖的方式促进近端可变5'剪接位点的利用。我们在SF2/ASF的N端RNA识别基序（RRM）和C端精氨酸/丝氨酸（RS）结构域中引入了选定的突变，并检测了所得重组蛋白的体外组成型和可变剪接以及与前体mRNA和mRNA的结合情况。在组成型剪接中无活性的突变体可影响可变剪接位点的选择，这表明这些活性涉及不同的分子相互作用。SF2/ASF RRM的RNP-1亚基序中由苯丙氨酸56和苯丙氨酸58介导的特定蛋白质-RNA接触对于组成型剪接至关重要，尽管RRM介导的与前体mRNA的结合并不需要它们。RS结构域对于组成型剪接活性也是必需的，精氨酸和丝氨酸残基都很重要。结构域缺失突变体的分析表明，RRM与一个中央简并RRM重复序列在结合RNA方面具有很强的协同作用。在没有RS结构域的情况下，这两个结构域足以实现可变剪接活性。
