Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing.
Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing.
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DOI：
10.1261/rna.041202.113
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发表时间：
2014-03
期刊：
影响因子：
--
通讯作者：
Deshimaru M
中科院分区：
文献类型：
--
作者：
Fukuda M;Kurihara K;Yamaguchi S;Oyama Y;Deshimaru M
腺苷到肌苷RNA编辑是调节各种生物过程的内源性机制。靶RNA的位点特异性和编辑状态依赖性降解方法可能是分析RNA编辑机制和调节生物过程的有力工具。在本文中，我们描述了一种策略，用于构建一个反式作用锤头状核酶，特异性切割靶RNA依赖于在特定位点的编辑状态。腺苷转肌苷（Adenosine-to-inosine，A-to-I）RNA编辑是一种参与多种生物学过程的内源性调控机制。靶RNA的位点特异性、编辑状态依赖性降解可能是分析RNA编辑机制和调节生物过程的有力工具。在此之前，我们设计了一种人工锤头状核酶（HHR），用于依赖于A到I RNA编辑状态的选择性、位点特异性RNA切割。在目前的工作中，我们开发了一种改进的策略，用于构建一种反式作用的HHR，该HHR特异性地切割腺苷而不是肌苷状态中的靶编辑位点。通过利用编码天然HHR的内在切割特异性的序列来实现对未编辑位点的特异性。我们在HHR文库中使用体外选择方法来选择含有三级稳定基序的延伸HHR，所述三级稳定基序促进HHR折叠成活性构象。利用这种方法，我们成功地构建了具有非编辑特异性切割的高活性HHR。此外，使用HHR切割，然后直接测序，我们证明了这种核酶可以切割从小鼠脑中提取的5-羟色胺2C受体（HTR 2C）mRNA，这取决于位点特异性编辑状态。这种未编辑的特异性切割也使我们能够通过使用直接测序同时定量多个位点的编辑率来分析E和C位点的编辑状态对其他位点的编辑的影响。我们的方法有可能阐明底物RNA中具有多个编辑位点的不同编辑状态相互依赖的机制。
Adenosine-to-inosine RNA editing is an endogenous mechanism for regulating various biological processes. A method for site-specific and editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. In this paper, we describe a strategy for constructing a trans-acting hammerhead ribozyme that specifically cleaves target RNA dependent on the editing state at the specific site. Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites.
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