Clarifying mammalian RISC assembly in vitro

Autor: Barry G. Garchow, Grace S. Tan, David Metzler, Marianthi Kiriakidou, Xuhang Liu
Rok vydání: 2011
Předmět:
Zdroj: BMC Molecular Biology
BMC Molecular Biology, Vol 12, Iss 1, p 19 (2011)
ISSN: 1471-2199
DOI: 10.1186/1471-2199-12-19
Popis: 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.
Databáze: OpenAIRE
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