An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein
An intronic splicing silencer causes skipping of the IIIb exon of fibroblast growth factor receptor 2 through involvement of polypyrimidine tract binding protein
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DOI：
10.1128/mcb.20.19.7388-7400.2000
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发表时间：
2000-10-01
影响因子：
5.3
通讯作者：
Garcia-Blanco, MA
中科院分区：
文献类型：
--
作者：
Carstens, RP;Wagner, EJ;Garcia-Blanco, MA
成纤维细胞生长因子受体2 （FGF-R2）转录本的选择性剪接涉及到外显子IIIb和IIIc的互斥使用，以产生两种不同的受体亚型。大鼠前列腺癌DT3细胞中外显子IIIb的适当剪接需要先前描述的顺式元件（ISAR，即“内含子剪接激活因子和阻遏因子”），该元件抑制外显子IIIc的剪接并激活外显子IIIb的剪接。该元件在大鼠前列腺AT3细胞中无功能，其抑制外显子IIIb的包含和外显子IIIc的剪接。我们现在已经确定了外显子IIIb上游的一个内含子元件，该元件导致外显子IIIb剪接的抑制。该元件的缺失使DT3细胞中外显子IIIb的剪接不需要ISAR，并导致AT3细胞中外显子mb的不适当包含。该元件由两个内含子剪接沉默子（LSS）序列ISS1和ISS2组成。ISS1序列嘧啶丰富，体外交联研究表明多嘧啶束结合蛋白（PTB）与该元素结合。竞争研究表明，ISS1中消除PTB结合的突变在体内减轻了剪接抑制。用含有外显子IIIb和内含子剪接沉默元件的minigene共转染PTB-1表达载体显示ptb介导的外显子IIIb剪接抑制。此外，所有描述的PTB亚型都同样能够介导这种效应。我们的研究结果支持剪接调节模型，其中外显子IIIc剪接不代表默认的剪接途径，而是一个外显子IIIb剪接的主动抑制发生在两个细胞中，并且DT3细胞能够克服这种抑制以剪接外显子IIIb。
Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for "intronic splicing activator and repressor") which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. We have now identified an intronic element upstream of exon IIIb that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon mb in AT3 cells. This element consists of two intronic splicing silencer (LSS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms were equally capable of mediating this effect. Our results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both tells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb.