Lavigueur A, La Branche H, Kornblihtt A R, Chabot B
Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Genes Dev. 1993 Dec;7(12A):2405-17. doi: 10.1101/gad.7.12a.2405.
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.
在HeLa细胞中包含270个核苷酸的人纤连蛋白ED1外显子需要存在一个位于中央的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。使用杂交模型前体mRNA底物，我们表明SE元件显著刺激ED1的3'剪接位点的使用。缺失和替换分析确定刺激序列为一段富含嘌呤的9个核苷酸（GAAGAAGAC）。SE元件从外显子内的各个位置刺激剪接到ED1的3'剪接位点，除非放置在该3'剪接位点下游293个核苷酸之外。增强子的作用不限于ED1受体位点，因为SE元件刺激人β-珠蛋白剪接，并且在体外也诱导原核序列中3'剪接位点的使用。我们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必需的，从而允许U2 snRNP与分支位点序列进行更有效的相互作用。在竞争实验中，一种主要包含SE序列的RNA特异性地消除了SE元件的作用，表明因子结合增强子元件以介导剪接刺激。使用RNA迁移率变动分析，我们表明SR蛋白与SE元件特异性相互作用。我们的结果表明，位于SE元件中的外显子序列通过与结合到3'剪接位点的因子相互作用，在指定剪接位点识别中起关键作用。
