人类AGO2介导由miRNA和siRNA靶向的RNA切割。

Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.

作者信息

Meister Gunter, Landthaler Markus, Patkaniowska Agnieszka, Dorsett Yair, Teng Grace, Tuschl Thomas

机构信息

Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, Box 186, New York, NY 10021, USA.

出版信息

Mol Cell. 2004 Jul 23;15(2):185-97. doi: 10.1016/j.molcel.2004.07.007.

DOI:10.1016/j.molcel.2004.07.007

PMID:15260970

Abstract

Argonaute proteins associate with small RNAs that guide mRNA degradation, translational repression, or a combination of both. The human Argonaute family has eight members, four of which (Ago1 through Ago4) are closely related and coexpressed in many cell types. To understand the biological function of the different Ago proteins, we set out to determine if Ago1 through Ago4 are associated with miRNAs as well as RISC activity in human cell lines. Our results suggest that miRNAs are incorporated indiscriminately of their sequence into Ago1 through Ago4 containing microRNPs (miRNPs). Purification of the FLAG/HA-epitope-tagged Ago containing complexes from different human cell lines revealed that endonuclease activity is exclusively associated with Ago2. Exogenously introduced siRNAs also associate with Ago2 for guiding target RNA cleavage. The specific role of Ago2 in guiding target RNA cleavage was confirmed independently by siRNA-based depletion of individual Ago members in combination with a sensitive positive-readout reporter assay.

摘要

AGO蛋白与引导mRNA降解、翻译抑制或两者兼有的小RNA结合。人类AGO蛋白家族有八个成员，其中四个成员（AGO1至AGO4）密切相关且在许多细胞类型中共同表达。为了了解不同AGO蛋白的生物学功能，我们着手确定AGO1至AGO4是否与miRNA以及人类细胞系中的RISC活性相关。我们的结果表明，miRNA被无差别地整合到包含AGO1至AGO4的微小RNA核蛋白复合物（miRNP）中。从不同人类细胞系中纯化带有FLAG/HA表位标签的AGO复合物，结果显示内切核酸酶活性仅与AGO2相关。外源导入的小干扰RNA（siRNA）也与AGO2结合以引导靶RNA切割。通过基于siRNA的单个AGO成员的敲除结合灵敏的正读出报告基因检测，独立证实了AGO2在引导靶RNA切割中的特定作用。