Trypanosome RNA editing: Simple guide RNA features enhance U deletion 100-fold
Trypanosome RNA editing: Simple guide RNA features enhance U deletion 100-fold
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DOI：
10.1128/mcb.21.3.884-892.2001
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发表时间：
2001-02-01
影响因子：
5.3
通讯作者：
Sollner-Webb, B
中科院分区：
文献类型：
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作者：
Cruz-Reyes, J;Zhelonkina, A;Sollner-Webb, B
锥虫RNA编辑是在反式作用引导RNA（gRNA）指导下通过U缺失和U插入对mRNA进行的大规模加工。已经使用合成的ATP酶（A6）前mRNA和gRNA在体外再现了U缺失循环和U插入循环。在这里，我们检查哪些gRNA特征对于这种U缺失是重要的。我们发现，最重要的是，这种编辑严重依赖于一些gRNA和一些mRNA残基邻接的锚双链体的单链特征，这是以前没有认识到的特征。这加上任何碱基配对序列来系链上游mRNA是所有gRNA需要使用纯化的编辑复合物或整个提取物来指导出乎意料地有效的体外U缺失。事实上，我们优化的gRNA构建体比天然gRNA更有效地支持忠实的U缺失高达100倍，并且它们可以编辑大多数mRNA分子。这是体外Il缺失的显著改进，其中先前的人工gRNA不比天然gRNA更有活性，并且编辑效率至多为百分之几。此外，这种编辑不受大多数其他先前提到的gRNA特征的刺激，包括其潜在的连接桥、3' OH部分、系链中的任何U残基、中心区域的保守结构或通常结合这些区域的蛋白质。我们的数据也对RNA编辑中活跃的进化力量有影响。
Trypanosome RNA editing is a massive processing of mRNA by U deletion and U insertion, directed by trans-acting guide RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6) pre-mRNA and gRNA. Here we examine which gRNA features are important for this U deletion. We find that, foremost, this editing depends critically on the single-stranded character of a few gRNA and a few mRNA residues abutting the anchor duplex, a feature not previously appreciated. That plus any base-pairing sequence to tether the upstream mRNA are all the gRNA needs to direct unexpectedly efficient in vitro U deletion, using either the purified editing complex or whole extract. In fact, our optimized gRNA constructs support faithful U deletion up to 100 times more efficiently than the natural gRNA, and they can edit the majority of mRNA molecules. This is a marked improvement of in vitro Il deletion, in which previous artificial gRNAs were no more active than natural gRNA and the editing efficiencies were at most a few percent. Furthermore, this editing is not stimulated by most other previously noted gRNA features, including its potential ligation bridge, 3' OH moiety, any U residues in the tether, the conserved structure of the central region, or proteins that normally bind these regions. Our data also have implications about evolutionary forces active in RNA editing.