A role for human Dicer in pre-RISC loading of siRNAs

Kumi Sakurai, Mohammed Amarzguioui, Dong-Ho Kim, Jessica Alluin, Bret Heale, Min-sun Song, Anne Gatignol, Mark A Behlke, John J Rossi

Research output: Contribution to journalArticlepeer-review

Abstract

RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.
Original languageEnglish
Pages (from-to)1510-25
Number of pages16
JournalNucleic Acids Research
Volume39
Issue number4
DOIs
Publication statusPublished - Mar 2011

Keywords

  • Argonaute Proteins
  • Cell Extracts
  • Eukaryotic Initiation Factor-2
  • HCT116 Cells
  • HEK293 Cells
  • Humans
  • RNA Interference
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • RNA-Induced Silencing Complex
  • Ribonuclease III

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