来自美国斯坦福大学儿科和遗传学系的 Mark A. Kay实验室发现在体内shRNAs和pre-miRNAs环状结构的位置对于Dicer加工准确性具有重要作用。

-2012年11月9日《细胞》


中文翻译


【题目】在体内shRNAs和pre-miRNAs的环状结构的位置对于Dicer加工准确性具有重要作用

【译文】ShRNA诱导的RNAi常用于生物探索和疾病治疗。Dicer的作用是使得miRNAs从前体中加工而成为内源性miRNAs,它可以使得shRNAs成为具有不同生物活性的siRNAs,这会影响到关闭寻靶作用的准确性和潜力。我们发现在细胞中Dicer在应切割位点周围诱导不精确的切割,这些位点是基于5′/3′计数原则。当切割位点位于环状结构2 nt处时,这些不正确的切割被消除。有趣的是,我们发现三分之一的哺乳动物内源性pre-miRNAs具有这些结构,而这些结构比Dicer产生的切割更加准确。为了实现“环状结构计数原则”,我们设计了一个可以大量减少关闭寻靶作用的反-HCV shRNAs。我们的结果证明了Dicer可以识别环状结构以促进加工的准确性,除了位于shRNAs/pre-miRNAs尾部的结构。我们的这些发现对于miRNA加工和未来shRNAs基于RNAi遗传筛选和治疗的设计具有重要意义。

英文原稿


[Title]The Loop Position of shRNAs and Pre-miRNAs Is Critical for the Accuracy of Dicer Processing In Vivo

[Authors]Shuo Gu, Lan Jin, Yue Zhang, Yong Huang, Feijie Zhang, Paul N. Valdmanis, Mark A. Kay

[Abstract]Short hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that, in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5′/3′ counting rules. These promiscuous noncanonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ∼1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a “loop-counting rule,” we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.

原文地址

http://www.cell.com/abstract/S0092-8674(12)01245-7

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