Precursor microRNA-programmed silencing complex assembly pathways in mammals

Mol Cell. 2012 May 25;46(4):507-17. doi: 10.1016/j.molcel.2012.03.010. Epub 2012 Apr 12.

Abstract

Assembly of microRNA ribonucleoproteins (miRNPs) or RNA-induced silencing complexes (RISCs) is essential for the function of miRNAs and initiates from processing of precursor miRNAs (pre-miRNAs) by Dicer or by Ago2. Here, we report an in vitro miRNP/RISC assembly assay programmed by pre-miRNAs from mammalian cell lysates. Combining in vivo studies in Dicer Knockout cells reconstituted with wild-type or catalytically inactive Dicer, we find that the miRNA loading complex (miRLC) is the primary machinery linking pre-miRNA processing to miRNA loading. We show that a miRNA precursor deposit complex (miPDC) plays a crucial role in Dicer-independent miRNA biogenesis and promotes miRNP assembly of certain Dicer-dependent miRNAs. Furthermore, we find that 5'-uridine, 3'-mid base pairing, and 5'-mid mismatches within pre-miRNAs promote their assembly into miPDC. Our studies provide a comprehensive view of miRNP/RISC assembly pathways in mammals, and our assay provides a versatile platform for further mechanistic dissection of such pathways in mammals.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Argonaute Proteins / chemistry
  • Argonaute Proteins / genetics
  • Argonaute Proteins / metabolism
  • Base Sequence
  • Cell Line
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / deficiency
  • DEAD-box RNA Helicases / genetics
  • DEAD-box RNA Helicases / metabolism
  • In Vitro Techniques
  • Mice
  • Mice, Knockout
  • MicroRNAs / chemistry
  • MicroRNAs / genetics*
  • MicroRNAs / metabolism*
  • Models, Biological
  • Protein Multimerization
  • RNA Processing, Post-Transcriptional
  • RNA-Induced Silencing Complex / chemistry
  • RNA-Induced Silencing Complex / genetics*
  • RNA-Induced Silencing Complex / metabolism*
  • Ribonuclease III / chemistry
  • Ribonuclease III / deficiency
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism

Substances

  • Ago2 protein, mouse
  • Argonaute Proteins
  • MicroRNAs
  • RNA-Induced Silencing Complex
  • Dicer1 protein, mouse
  • Ribonuclease III
  • DEAD-box RNA Helicases