Slicer function of Drosophila Argonautes and its involvement in RISC formation
Slicer function of Drosophila Argonautes and its involvement in RISC formation
复制标题
DOI：
10.1101/gad.1370605
复制
发表时间：
2005-12-01
影响因子：
10.5
通讯作者：
Siomi, MC
中科院分区：
文献类型：
--
作者：
Miyoshi, K;Tsukumo, H;Siomi, MC
Argonaute蛋白在RNA沉默中发挥重要而独特的作用。人类Argonaute 2（hAgo2）显示负责RNA干扰（RNAi）中的靶RNA切割（"Slicer"）活性，而其他Argonaute亚家族成员在人类中不表现出Slicer活性。在果蝇中，AGO 2被证明具有Slicer活性。在这里，我们表明，AGO 1，另一个成员的果蝇Argonaute亚家族，免疫纯化的施耐德2（S2）细胞与microRNA（miRNA）和切割目标RNA完全互补的miRNA。用重组全长AGO1重建切割酶活性。因此，与人类不同，在果蝇中，AGO 1和AGO 2都具有Slicer功能。此外，用AGO1和AGO2的重组PIWI结构域重建Slicer活性证明Argonaute中的其他区域对于小干扰RNA（siRNA）结合和切割活性不是严格必需的。已经表明，在AGO2缺乏的情况下，siRNA双链体不会解旋，因此不会形成RNA诱导的沉默复合物（RISC）。我们表明，在S2裂解液中加入siRNA双链体后，乘客链以AGO2依赖性方式裂解，乘客链的核酸酶抗性修饰损害RISC形成。这些发现产生了一种新的模型，其中AGO2作为siRNA双链体的乘客链的"切片器"直接参与RISC形成。
Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ("Slicer") activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here we show that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. We show that upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as "Slicer" of the passenger strand of the siRNA duplex.