FUNCTIONAL-ANALYSIS OF PREMESSENGER RNA SPLICING FACTOR SF2/ASF STRUCTURAL DOMAINS
FUNCTIONAL-ANALYSIS OF PREMESSENGER RNA SPLICING FACTOR SF2/ASF STRUCTURAL DOMAINS
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DOI：
10.1002/j.1460-2075.1993.tb06160.x
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发表时间：
1993-12-01
期刊：
影响因子：
11.4
通讯作者：
KRAINER, AR
中科院分区：
文献类型：
--
作者：
CACERES, JF;KRAINER, AR
人mrna前剪接因子SF2/ASF在体外具有一般剪接所需的活性，并通过反对hnRNP A1以浓度依赖性的方式促进近端替代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）结构域引入了选择的突变，并分析了产生的重组蛋白在体外的组成剪接和选择性剪接以及与pre-mRNA和mRNA的结合。在组成剪接中失活的突变体可以影响选择性剪接位点的选择，表明这些活动涉及不同的分子相互作用。在SF2/ASF RRM的RNP-1子基序中，由Phe56和Phe58介导的特异性蛋白- RNA接触对于组成剪接是必不可少的，尽管它们不是RRM介导的与pre-mRNA结合所必需的。RS结构域也是组成剪接活性所必需的，Arg和Ser残基都很重要。对结构域缺失突变体的分析表明，RRM和一个中心退化的RRM重复序列在结合RNA时具有很强的协同作用。在没有RS结构域的情况下，这两个结构域足以进行备选剪接活动。
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 contacts 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.