High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2
High-Throughput Analysis Reveals Rules for Target RNA Binding and Cleavage by AGO2
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DOI：
10.1016/j.molcel.2019.06.012
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发表时间：
2019-08-22
期刊：
影响因子：
16
通讯作者：
Greenleaf, William J.
中科院分区：
文献类型：
--
作者：
Becker, Winston R.;Ober-Reynolds, Benjamin;Greenleaf, William J.
Argonaute蛋白装载有microRNA（miRNA）或小干扰RNA（siRNA），形成RNA诱导的沉默复合物（RISC），抑制靶RNA表达。由于对RISC结合和切割的序列决定因素的不完全理解，预测miRNA和siRNA的生物学靶点、特异性和效率受到阻碍。我们应用高通量的方法来测量的关联动力学，平衡结合能，和单营业额小鼠AGO2 RISC的裂解率。我们发现，RISC很容易容忍插入多达7个核苷酸在其目标相反的中心区域的指导。我们的数据揭示了特定的指南：靶错配，提高靶切割的速率，提出了新的siRNA设计策略。使用这些数据，我们推导出RISC结合和靶切割的定量模型，并表明我们的体外测量和模型预测在工程细胞系统中的敲除。
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.