Gene Suppression Laboratory, Department of Plant and Soil Sciences and Kentucky Tobacco and Research Development Center, University of Kentucky, Lexington, Kentucky 40546, USA.
Plant Cell. 2012 Feb;24(2):415-27. doi: 10.1105/tpc.111.094144. Epub 2012 Feb 17.
MicroRNAs (miRNAs) and other endogenous small RNAs act as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. The interrogation of small RNA functions requires an effective, widely applicable method to specifically block small RNA function. Here, we report the development of a highly effective technology that targets specific endogenous miRNAs or small interfering RNAs for destruction in Arabidopsis thaliana. We show that the expression of a short tandem target mimic (STTM), which is composed of two short sequences mimicking small RNA target sites, separated by a linker of an empirically determined optimal size, leads to the degradation of targeted small RNAs by small RNA degrading nucleases. The efficacy of the technology was demonstrated by the strong and specific developmental defects triggered by STTMs targeting three miRNAs and an endogenous siRNA. In summary, we developed an effective approach for the destruction of endogenous small RNAs, thereby providing a powerful tool for functional genomics of small RNA molecules in plants and potentially animals.
MicroRNAs (miRNAs) 和其他内源性小 RNA 作为真核生物基因组、转录组和蛋白质组的序列特异性调控因子发挥作用。小 RNA 功能的研究需要一种有效且广泛适用的方法来特异性阻断小 RNA 的功能。在这里，我们报告了一种针对拟南芥中特定内源性 miRNAs 或小干扰 RNA 进行破坏的高效技术的开发。我们表明，短串联靶标模拟物 (STTM) 的表达，由两个短序列组成，模拟小 RNA 靶标位点，由经验确定的最佳大小的接头隔开，导致靶向小 RNA 被小 RNA 降解核酶降解。通过靶向三个 miRNA 和一个内源性 siRNA 的 STTM 触发的强烈和特异性发育缺陷证明了该技术的功效。总之，我们开发了一种有效破坏内源性小 RNA 的方法，从而为植物中小 RNA 分子的功能基因组学提供了一种强大的工具，并且可能在动物中也有应用。
