title: Clarifying mammalian RISC assembly in vitro.
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.
authors.affiliation: Department of Medicine, Division of Rheumatology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
authors.forename: Grace S
authors.initials: GS
authors.lastname: Tan
authors.forename: Barry G
authors.initials: BG
authors.lastname: Garchow
authors.forename: Xuhang
authors.initials: X
authors.lastname: Liu
authors.forename: David
authors.initials: D
authors.lastname: Metzler
authors.forename: Marianthi
authors.initials: M
authors.lastname: Kiriakidou
mesh_terms: D000818:Animals; D060565:Argonaute Proteins; D001483:Base Sequence; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D034743:RNA-Induced Silencing Complex; D011994:Recombinant Proteins; D012313:RNA; D043244:Ribonuclease III