Structural and biochemical insights into the dicing mechanism of mouse Dicer: A conserved lysine is critical for dsRNA cleavage

Zhihua Du, John K. Lee, Richard Tjhen, Robert M. Stroud, Thomas L. James

Research output: Contribution to journalArticle

58 Scopus citations

Abstract

Dicer, an RNase III enzyme, initiates RNA interference by processing precursor dsRNAs into mature microRNAs and small-interfering RNAs. It is also involved in loading and activation of the RNA-induced silencing complex. Here, we report the crystal structures of a catalytically active fragment of mouse Dicer, containing the RNase IIIb and dsRNA binding domains, in its apo and Cd2+-bound forms, at 1.68- and 2.8-Å resolution, respectively. Models of this structure with dsRNA reveal that a lysine residue, highly conserved in Dicer RNase IIIa and IIIb domains and in Drosha RNase IIIb domains, has the potential to participate in the phosphodiester bond cleavage reaction by stabilizing the transition state and leaving group of the scissile bond. Mutational and enzymatic assays confirm the importance of this lysine in dsRNA cleavage, suggesting that this lysine represents a conserved catalytic residue of Dicers. The structures also reveals a ≈45-aa region within the RNase IIIb domain that harbors an α-helix at the N-terminal half and a flexible loop at the C-terminal half, features not present in previously reported structures of homologous RNase III domains from either bacterial RNase III enzymes or Giardia Dicer. N-terminal residues of this α-helix have the potential to engage in minor groove interaction with dsRNA substrates.

Original languageEnglish (US)
Pages (from-to)2391-2396
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number7
DOIs
StatePublished - Feb 19 2008

Keywords

  • RNA interference
  • RNase III
  • X-ray crystallography
  • dsRNA processing

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