Binding of equine infectious anemia virus Rev to an exon splicing enhancer mediates alternative splicing and nuclear export of vicar mRNAs
Binding of equine infectious anemia virus Rev to an exon splicing enhancer mediates alternative splicing and nuclear export of vicar mRNAs
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DOI：
10.1128/mcb.20.10.3550-3557.2000
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发表时间：
2000-05-01
影响因子：
5.3
通讯作者：
Carpenter, S
中科院分区：
文献类型：
--
作者：
Belshan, M;Park, GS;Carpenter, S
除了促进不完全剪接的病毒mRNA的核输出外，马传染性贫血病毒（EIAV）Rev还调节达特/rev mRNA第三外显子的选择性剪接。在Rev存在的情况下，双顺反子RNA的该外显子在一部分剪接的mRNA中被跳过。在本报告中，外显子3使用的顺式作用要求与Rev结合和转运不完全剪接RNA所需的序列相关。富含嘌呤的外显子剪接增强子（ESE）的存在是外显子3识别所必需的，Rev的加入抑制了外显子3的剪接。谷胱甘肽-S-转移酶（GST）-Rev结合到含有ESE的探针上，并且ESE内GAA重复序列突变为GCA抑制RNA剪接实验中外显子3的识别和体外GST-Rev结合。这些结果表明，Rev通过在ESE处或附近结合来阻断SR蛋白-ESE相互作用，从而调节可变剪接。含有ESE的57个核苷酸序列足以介导不完全剪接RNA的Rev依赖性核输出。Rev输出活性显著抑制突变的。ESE或通过与SF 2/ASF反式互补。这些结果表明，ESE作为Rev反应元件发挥功能，并证明EIAV Rev通过蛋白质-RNA相互作用介导外显子3排除，这是不完全剪接的病毒RNA有效输出所需的。
In addition to facilitating the nuclear export of incompletely spliced viral mRNAs, equine infectious anemia virus (EIAV) Rev regulates alternative splicing of the third exon of the tat/rev mRNA. In the presence of Rev, this exon of the bicistronic RNA is skipped in a fraction of the spliced mRNAs. In this report, the cis-acting requirements for exon 3 usage were correlated with sequences necessary for Rev binding and transport of incompletely spliced RNA. The presence of a purine-rich exon splicing enhancer (ESE) was required for exon 3 recognition, and the addition of Rev inhibited exon 3 splicing. Glutathione-S-transferase (GST)-Rev bound to probes containing the ESE, and mutation of GAA repeats to GCA within the ESE inhibited both exon 3 recognition in RNA splicing experiments and GST-Rev binding in vitro. These results suggest that Rev regulates alternative splicing by binding at or near the ESE to block SR protein-ESE interactions. A 57-nucleotide sequence containing the ESE was sufficient to mediate Rev-dependent nuclear export of incompletely spliced RNAs. Rev export activity was significantly inhibited by mutation of the. ESE or by trans-complementation with SF2/ASF. These results indicate that the ESE functions as a Rev-responsive element and demonstrate that EIAV Rev mediates exon 3 exclusion through protein-RNA interactions required for efficient export of incompletely spliced viral RNAs.