Abstract
BackgroundArgonaute, 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.ResultsThe 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.ConclusionsOur 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.
Highlights
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
Inclusion of TAR RNA binding protein (TRBP) to the reconstitution reactions did not affect RISC activity (Figure 2A). These results indicate that Argonaute 2 (Ago2) and Dicer alone were sufficient for canonical RISC loading in vitro
Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC
Summary
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) assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active in vitro and constitute non-canonical RISCs. MicroRNAs (miRNAs) are small (~22 nucleotide) noncoding RNAs that associate with Argonaute proteins in ribonucleoprotein complexes (miRNPs or RISCs) [1,2,3,4,5]. Post-transcriptional nuclear processing of primary miRNA transcripts (pri-miRNAs) by the RNAse III enzyme Drosha and its RNA-binding partner, DiGeorge syndrome Critical Region gene 8 protein (DGCR8) [15,16,17,18], generates ~ 65-75 nucleotide (nt) hairpin-structured miRNA precursors (pre-miRNAs). The mechanisms involved in miRNA strand selection in mammals are poorly understood
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