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Open Access Highly Accessed Research article

Clarifying mammalian RISC assembly in vitro

Grace S Tan1, Barry G Garchow1, Xuhang Liu2, David Metzler1 and Marianthi Kiriakidou1*

Author Affiliations

1 Department of Medicine, Division of Rheumatology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

2 Department of Pathology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

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BMC Molecular Biology 2011, 12:19  doi:10.1186/1471-2199-12-19

Published: 29 April 2011

Abstract

Background

Argonaute, the core component of the RNA induced silencing complex (RISC), binds to mature miRNAs and regulates gene expression at transcriptional or post-transcriptional level. We recently reported that Argonaute 2 (Ago2) also assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active in vitro and constitute non-canonical RISCs.

Results

The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates in vitro RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate in vitro pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes.

Conclusions

Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.