Exon inclusion is dependent on predictable exonic splicing enhancers
Exon inclusion is dependent on predictable exonic splicing enhancers
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DOI：
10.1128/mcb.25.16.7323-7332.2005
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发表时间：
2005-08-01
影响因子：
5.3
通讯作者：
Chasin, LA
中科院分区：
文献类型：
--
作者：
Zhang, XHF;Kangsamaksin, T;Chasin, LA
我们之前已经基于人类外显子和非外显子序列的统计比较，制定了一个大约2,000种RNA八聚体的列表，作为推定的外显子剪接增强子（PESE）（X. H. Zhang和L. A. Chasin，Genes Dev. 18：1241-1250，2004）。当插入到一个不良剪接的测试外显子，所有八个测试的八聚体刺激剪接，结果与他们的鉴定为外显子剪接增强子（ESEs）一致。在这里，我们提出了一个更严格的测试的有效性，这份名单的PESE。二十二个自然发生的例子，非重叠PESE或PESE集群被确定在六个哺乳动物外显子，五个测试的六个外显子组成型剪接。22个单独的PESE或PESE簇中的每一个都通过定点诱变（通常通过单碱基取代）被破坏。22个中断中的18个（82%）导致剪接效率降低。相比之下，24个对照突变对剪接的影响很小或没有影响。这种高成功率表明，大多数PESE在其自然环境中发挥着ESE的作用。像大多数外显子一样，这些外显子包含几个PESE。由于敲除几个中的任何一个都可能使剪接效率降低几倍，因此我们得出结论，外显子中的ESE之间几乎没有冗余，它们必须协同工作以优化剪接。
We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.