A short sequence within two purine-rich enhancers determines 5′ splice site specificity
A short sequence within two purine-rich enhancers determines 5′ splice site specificity
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DOI：
10.1128/mcb.18.1.343
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发表时间：
1998-01-01
影响因子：
5.3
通讯作者：
Berget, SM
中科院分区：
文献类型：
--
作者：
Elrick, LL;Humphrey, MB;Berget, SM
富含嘌呤的增强子是通常通过激活弱上游3'剪接位点来促进包含选择性外显子的外显子序列。最近描述的来自钙调蛋白基因的富含嘌呤的增强子具有额外的活性，通过该活性它指导选择性外显子内的竞争性5'剪接位点。我们已经比较了钙调蛋白基因增强子与来自鸡心肌肌钙蛋白T（cTNT）基因的另一种富含嘌呤的增强子调节侧翼剪接位点的能力，尽管序列和长度相似，当置于内部外显子中的竞争性5 ′剪接位点之间时，这两种增强子表现出对5 ′剪接位点选择的显著不同的特异性。32个核苷酸的钙调蛋白基因增强子引起外显子-内部5 ′剪接位点的有效利用，而30个核苷酸的cTNT增强子引起外显子-末端5 ′剪接位点的有效利用，两种增强子介导的剪接途径都代表了默认途径的调节，其中利用了两个5'剪接位点。每个增强子是多部分的，由两个富含嘌呤的简单（GAR）（n）重复序列与两个增强子特异性序列交叉组成，整个增强子对于最大剪接位点选择性是必需的;然而，从每个增强子的3'端起的5-至7-核苷酸区域决定剪接位点选择性。将两个增强子的这个短区域互换的突变改变了特异性。cTNT增强子中决定5'剪接位点选择性的部分与显示出对3'剪接位点的活化最重要的部分不同，表明增强子环境可对活性具有主要影响。这些结果是第一个表明单个富含嘌呤的增强子可区分侧翼剪接位点。此外，剪接位点选择的特异性定位于两个增强子内的短区域，表明增强子序列的细微差异可对剪接途径产生深远的影响。
Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3' splice sites, A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5' splice sites within an alternative exon, In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites, Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5' splice site choice when placed between competing 5' splice sites in an internal exon, The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5' splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5' splice site, Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5' splice sites were utilized, Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)(n) repeat interdigitated with two enhancer-specific sequences, The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3' end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5' splice site selectivity was different than that shown to be maximally important for activation of a 3' splice site, suggesting that enhancer environment can have a major impact on activity, These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites, Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.