高通量分析揭示了 AGO2 对靶 RNA 结合和切割的规则。
High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2.
机构信息
Program in Biophysics, Stanford University, Stanford, CA 94305, USA.
Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA.
出版信息
Mol Cell. 2019 Aug 22;75(4):741-755.e11. doi: 10.1016/j.molcel.2019.06.012. Epub 2019 Jul 16.
Argonaute proteins loaded with microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC), which represses target RNA expression. Predicting the biological targets, specificity, and efficiency of both miRNAs and siRNAs has been hamstrung by an incomplete understanding of the sequence determinants of RISC binding and cleavage. We applied high-throughput methods to measure the association kinetics, equilibrium binding energies, and single-turnover cleavage rates of mouse AGO2 RISC. We find that RISC readily tolerates insertions of up to 7 nt in its target opposite the central region of the guide. Our data uncover specific guide:target mismatches that enhance the rate of target cleavage, suggesting novel siRNA design strategies. Using these data, we derive quantitative models for RISC binding and target cleavage and show that our in vitro measurements and models predict knockdown in an engineered cellular system.
Argonaute 蛋白与 microRNAs (miRNAs) 或小干扰 RNA (siRNAs) 结合形成 RNA 诱导沉默复合物 (RISC)，从而抑制靶 RNA 的表达。由于对 RISC 结合和切割的序列决定因素缺乏完整的了解，miRNAs 和 siRNAs 的生物靶标、特异性和效率的预测一直受到阻碍。我们应用高通量方法来测量小鼠 AGO2 RISC 的结合动力学、平衡结合能和单轮切割速率。我们发现 RISC 很容易容忍其靶标中与中心区域相对的中央区域插入长达 7 个核苷酸。我们的数据揭示了特定的引导：靶标错配，可提高靶标切割的速率，这表明了新的 siRNA 设计策略。利用这些数据，我们推导出 RISC 结合和靶标切割的定量模型，并表明我们的体外测量和模型可以预测工程细胞系统中的基因敲低。
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