The importance of RNA structure in RNA editing and a potential proofreading mechanism for correct guide RNA:pre-mRNA binary complex formation
The importance of RNA structure in RNA editing and a potential proofreading mechanism for correct guide RNA:pre-mRNA binary complex formation
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DOI：
10.1016/j.jmb.2006.03.041
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发表时间：
2006-06-09
影响因子：
5.6
通讯作者：
Hajduk, Stephen L.
中科院分区：
文献类型：
--
作者：
Golden, Daniel E.;Hajduk, Stephen L.
锥虫体内的RNA编辑是一个转录后过程，负责纠正许多线粒体mrna的编码序列。尿苷碱基通过酶联反应从mRNA中特异性地添加或删除，其中预先编辑的mRNA被特异性地切割，尿苷碱基被添加或删除，校正的mRNA被连接。这个过程是由RNA分子指导的，称为引导RNA （gRNA）。这类小的非编码RNA在RNA编辑中发挥作用的能力对这些生物至关重要。通常，grna的转录独立于其同源mRNA，并退火形成二元RNA复合物。这个过程中的一个例外是细胞色素氧化酶亚基II (COII) mRNA，它在其3'非翻译区编码其gRNA。这种gRNA缺乏反式功能。使用体外编辑实验，我们发现通过增加沃森-克里克碱基配对来改善锚定区域的热力学稳定性足以赋予反编辑活性。我们进一步表明，在gRNA已知功能区域之外的点突变既会诱导gRNA的构象重排，又会导致编辑速率的降低。综上所述，这些结果使我们提出了gRNA形成过程中潜在的校对步骤的模型：预编辑的mRNA二元复合体。该机制依赖于通过沃森-克里克碱基配对提供给RNA复合物的热力学稳定性。通过gRNA的突变，我们证明了gRNA结构对RNA编辑反应的重要性。(c) 2006 Elsevier Ltd.版权所有。
RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridine bases are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is cleaved specifically, uridine bases are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, non-coding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of their cognate mRNA and anneal to form a binary RNA complex. An exception from this process is the cytochrome oxidase subunit II (COII) mRNA, which encodes its gRNA within its 3' untranslated region. This gRNA lacks the ability to function in trans. Using an in vitro editing assay, we find that improving thermodynamic stability to the anchor region through increased Watson-Crick base-pairing is sufficient to impart trans editing activity. We further show that a point mutation outside the known functional regions of a gRNA induces both a conformational rearrangement of the gRNA and causes a decrease in the rate of editing. Taken together, these results lead us to propose a model for a potential proofreading step in the formation of a gRNA:pre-edited mRNA binary complex. The mechanism relies on the thermodynamic stability supplied to the RNA complex through Watson-Crick base-pairing. Through mutations in the gRNA, we demonstrate the importance of gRNA structure to the RNA editing reaction. (c) 2006 Elsevier Ltd. All rights reserved.