Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing.
Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing.
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DOI：
10.1186/1471-2199-9-23
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发表时间：
2008-02-12
影响因子：
--
通讯作者：
Gaur, Rajesh K.
中科院分区：
文献类型：
--
作者：
Kim, Dong-Suk;Gusti, Veronica;Dery, Kenneth J.;Gaur, Rajesh K.
尽管在理解组成性剪接和选择性剪接机制方面取得了巨大进展，这是基因表达途径中沿着重要而广泛的一步，但我们在这一步有意调控基因表达的能力仍然很差。本研究旨在探讨茶碱依赖的剪接开关是否能调节RNA-茶碱复合物中的选择性剪接。我们构建了一系列前体mRNA，其中BPS插入茶碱适体中。我们发现，茶碱诱导的螯合BPS抑制前mRNA剪接在体外和体内以剂量依赖性的方式。一些证据表明，茶碱依赖的剪接抑制是高度特异性的，RNA-茶碱复合物的热力学稳定性以及BPS在该复合物中的位置影响剪接抑制的效率。最后，我们构建了一个选择性剪接模型pre-mRNA底物，其中茶碱引起外显子跳跃在体外和体内，这表明小分子RNA相互作用可以调节选择性剪接。这些发现提供了随意控制剪接模式的能力，对基础、生物技术和生物医学研究具有重要意义。
Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing. We constructed a series of pre-mRNAs in which the BPS was inserted within theophylline aptamer. We show that theophylline-induced sequestering of BPS inhibits pre-mRNA splicing both in vitro and in vivo in a dose-dependent manner. Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition. Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing. These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.
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