Kawamata Tomoko, Seitz Hervé, Tomari Yukihide
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Japan.
Nat Struct Mol Biol. 2009 Sep;16(9):953-60. doi: 10.1038/nsmb.1630. Epub 2009 Aug 16.
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.
微小RNA（miRNA）通过RNA诱导沉默复合体（RISC）调控其靶mRNA的表达，该复合体以AGO蛋白家族成员作为核心组分。在黑腹果蝇中，miRNA通常被分选到含有AGO1的RISC（AGO1-RISC）中。我们建立了一种天然凝胶系统，可从生化角度剖析AGO1-RISC的组装途径。我们发现，miRNA-miRNA双链以ATP依赖的方式作为双链RNA加载到AGO1中。相反，出乎意料的是，miRNA-miRNA双链的解旋是一个被动过程，不需要ATP或AGO1的核酸酶活性。中心错配将miRNA-miRNA*双链引导至前AGO1-RISC，而种子区或引导链第12-15位的错配则促进前AGO1-RISC转化为成熟的AGO1-RISC。我们的研究结果表明，miRNA的解旋是靶标识别的精确镜像过程，且这两个过程都反映了AGO蛋白中RNA的独特几何结构。
