A SPLICING ENHANCER IN THE HUMAN FIBRONECTIN ALTERNATE ED1-EXON INTERACTS WITH SR-PROTEINS AND STIMULATES U2-SNRNP BINDING
A SPLICING ENHANCER IN THE HUMAN FIBRONECTIN ALTERNATE ED1-EXON INTERACTS WITH SR-PROTEINS AND STIMULATES U2-SNRNP BINDING
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DOI：
10.1101/gad.7.12a.2405
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发表时间：
1993-12-01
影响因子：
10.5
通讯作者：
CHABOT, B
中科院分区：
文献类型：
--
作者：
LAVIGUEUR, A;LABRANCHE, H;CHABOT, B
在HeLa细胞中包含270个核苷酸的人纤连蛋白ED 1外显子需要存在位于中心的81个核苷酸的外显子序列。我们进行了一系列体外实验，旨在了解与这种剪接增强子（SE）相关的结构和功能特征。使用混合模型前mRNA底物，我们表明，SE元件显着刺激ED 1的3'剪接位点的使用。缺失和置换分析将刺激序列鉴定为富含嘌呤的9个核苷酸的延伸（GAAGAAGAC）。SE元件刺激从外显子内的不同位置剪接到ED 1 3'剪接位点，除了当位于该3'剪接位点下游293个核苷酸之外。增强子的作用不限于ED 1受体位点，因为SE元件刺激人β-珠蛋白剪接，并且还在体外诱导原核序列中3 ′剪接位点的使用。我们已经探索了纤连蛋白剪接增强子的作用机制，发现SE元件是早期剪接复合物有效组装所需的，从而使U2 snRNP与分支位点序列更有效地相互作用。在竞争实验中，主要含有SE序列的RNA特异性地消除了SE元件的作用，这表明因子结合增强子元件以介导剪接的刺激。使用RNA迁移率变动分析，我们表明，SR蛋白相互作用，特别是与SE元素。我们的研究结果表明，位于SE元件的外显子序列通过与3'剪接位点结合因子的相互作用在指定剪接位点识别中起着至关重要的作用。
The inclusion of the 270-nucleotide human fibronectin ED1 exon in HeLa cells requires the presence of a centrally located 81-nucleotide exon sequence. We have conducted a series of in vitro experiments aimed at understanding the structural and functional features associated with this splicing enhancer (SE). Using hybrid model pre-mRNA substrates, we show that the SE element markedly stimulates the use of the 3' splice site of ED1. Deletion and replacement analysis identifies the stimulating sequences as a purine-rich stretch of 9 nucleotides (GAAGAAGAC). The SE element stimulates splicing to the ED1 3' splice site from various positions within the exon except when placed beyond 293 nucleotides downstream from that 3' splice site. The action of the enhancer is not limited to the ED1 acceptor site because the SE element stimulates human beta-globin splicing and also induces the use of a 3' splice site in a prokaryotic sequence in vitro. We have explored the mechanism of action of the fibronectin splicing enhancer and found that the SE element is required for efficient assembly of early splicing complexes, allowing a more efficient interaction of the U2 snRNP with branch site sequences. In competition experiments, an RNA containing mainly SE sequences specifically abolished the action of the SE element, suggesting that factors bind the enhancer element to mediate stimulation of splicing. Using RNA mobility shift assays we show that SR proteins interact specifically with the SE element. Our results demonstrate that exon sequences lying in the SE element play a crucial role in specifying splice site recognition through interactions with factors binding to the 3' splice site.