Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA
Tra2-Mediated Recognition of HIV-1 5′ Splice Site D3 as a Key Factor in the Processing of vpr mRNA
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DOI：
10.1128/jvi.02756-12
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发表时间：
2013-03-01
影响因子：
5.4
通讯作者：
Schaal, Heiner
中科院分区：
文献类型：
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作者：
Erkelenz, Steffen;Poschmann, Gereon;Schaal, Heiner
小的非编码HIV-1前导外显子3由其剪接位点A2和D3定义。虽然3'剪接位点（3' ss）A2需要被激活以形成vpr mRNA，但下游内含子3内的vpr起始密码子的位置需要5' ss D3处的剪接沉默。在这里，我们表明，包括HIV-1外显子3和vpr mRNA的加工是促进外显子剪接增强子（ESEvpr）之间的外显子剪接沉默ESSV和5' SS D3定位。发现ESEvpr序列被Transformer 2（Tra 2）蛋白家族的成员结合。这些蛋白质在前病毒转染的细胞中的共表达导致外显子3包涵体水平的增加，证实它们通过ESEvpr起作用。进一步的分析显示，ESEvpr支持U1 snRNA在5' ss D3处的结合，允许跨越上游外显子与3' ss A2的桥接相互作用。与此一致，5' ss D3与U1 snRNA 5'端互补性的增加或减少伴随着vpr表达水平的升高或降低。然而，通过所提出的桥接相互作用激活3' ss A2并不依赖于5' ss D3的剪接能力，因为使其剪接缺陷但仍能有效结合U1 snRNA，从而保持了D3的增强功能。因此，我们提出，剪接在3' ss A2之间的时间发生U1 snRNA的结合和剪接在D3。
Small noncoding HIV-1 leader exon 3 is defined by its splice sites A2 and D3. While 3' splice site (3' ss) A2 needs to be activated for vpr mRNA formation, the location of the vpr start codon within downstream intron 3 requires silencing of splicing at 5' ss D3. Here we show that the inclusion of both HIV-1 exon 3 and vpr mRNA processing is promoted by an exonic splicing enhancer (ESEvpr) localized between exonic splicing silencer ESSV and 5' ss D3. The ESEvpr sequence was found to be bound by members of the Transformer 2 (Tra2) protein family. Coexpression of these proteins in provirus-transfected cells led to an increase in the levels of exon 3 inclusion, confirming that they act through ESEvpr. Further analyses revealed that ESEvpr supports the binding of U1 snRNA at 5' ss D3, allowing bridging interactions across the upstream exon with 3' ss A2. In line with this, an increase or decrease in the complementarity of 5' ss D3 to the 5' end of U1 snRNA was accompanied by a higher or lower vpr expression level. Activation of 3' ss A2 through the proposed bridging interactions, however, was not dependent on the splicing competence of 5' ss D3 because rendering it splicing defective but still competent for efficient U1 snRNA binding maintained the enhancing function of D3. Therefore, we propose that splicing at 3' ss A2 occurs temporally between the binding of U1 snRNA and splicing at D3.