The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo.
The loop position of shRNAs and pre-miRNAs is critical for the accuracy of dicer processing in vivo.
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DOI：
10.1016/j.cell.2012.09.042
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发表时间：
2012-11-09
期刊：
影响因子：
64.5
通讯作者：
Kay MA
中科院分区：
文献类型：
--
作者：
Gu S;Jin L;Zhang Y;Huang Y;Zhang F;Valdmanis PN;Kay MA
短发夹RNA（Short-hairpin RNA，shRNA）诱导的RNAi被用于生物学发现和治疗。Dicer的正常作用是从其前体中释放内源性miRNA，将shRNA加工成不同的生物活性siRNA，影响其功效和脱靶潜力。我们发现，在细胞中，Dicer基于先前描述的5′/3′计数规则在预期位点周围诱导不精确的切割事件。当切割位点位于离凸起或环2nt处时，这些混杂的非典型切割被废除。有趣的是，我们观察到约1/3的含有这种结构的哺乳动物内源性pre-miRNA被Dicer更精确地加工。实施新的“环计数规则”，我们设计了有效的抗HCV shRNA，大大降低了脱靶效应。我们的研究结果表明，Dicer除了识别shRNA/pre-miRNA的末端外，还识别环/凸起结构以进行准确加工。这对miRNA加工和未来设计用于基于RNAi的遗传筛选和治疗的shRNA具有重要意义。
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 non-canonical 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 new “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.
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