Structural determinants of miRNAs for RISC loading and slicer-independent unwinding
Structural determinants of miRNAs for RISC loading and slicer-independent unwinding
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DOI：
10.1038/nsmb.1630
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发表时间：
2009-09-01
影响因子：
16.8
通讯作者：
Tomari, Yukihide
中科院分区：
文献类型：
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作者：
Kawamata, Tomoko;Seitz, Herve;Tomari, Yukihide
MicroRNAs(MiRNAs)通过RNA诱导沉默复合体(RISC)调节其靶mRNAs的表达，RISC含有ArgAerte(AGO)家族蛋白作为核心成分。在果蝇中，miRNAs通常被分类为含有Ago1的RISC(Ago1-RISC)。我们建立了一个天然的凝胶系统，可以生化地剖析Ago1-RISC组装途径。我们发现miRNA-miRNA*双链以ATP依赖的方式以双链RNA的形式被加载到Ago1中。相反，出人意料的是，miRNA-miRNA*双链的解离是一个被动的过程，不需要ATP或Ago1的切片器活性。中心错配将miRNA-miRNA*双链直接转化为前Ago1-RISC，而种子或引导链位置12-15的错配促进了前Ago1-RISC向成熟Ago1-RISC的转化。我们的发现表明，miRNAs的解离是目标识别的精确镜像过程，这两个过程都反映了AGO蛋白质中RNAs的独特几何结构。
MicroRNAs (miRNAs) regulate expression of their target mRNAs through the RNA-induced silencing complex (RISC), which contains an Argonaute (Ago) family protein as a core component. In Drosophila melanogaster, miRNAs are generally sorted into Ago1-containing RISC (Ago1-RISC). We established a native gel system that can biochemically dissect the Ago1-RISC assembly pathway. We found that miRNA-miRNA* duplexes are loaded into Ago1 as double-stranded RNAs in an ATP-dependent fashion. In contrast, unexpectedly, unwinding of miRNA-miRNA* duplexes is a passive process that does not require ATP or slicer activity of Ago1. Central mismatches direct miRNA-miRNA* duplexes into pre-Ago1-RISC, whereas mismatches in the seed or guide strand positions 12-15 promote conversion of pre-Ago1-RISC into mature Ago1-RISC. Our findings show that unwinding of miRNAs is a precise mirror-image process of target recognition, and both processes reflect the unique geometry of RNAs in Ago proteins.