Structural determinants of RNA recognition and cleavage by Dicer

Nat Struct Mol Biol. 2007 Oct;14(10):934-40. doi: 10.1038/nsmb1293. Epub 2007 Sep 16.

Abstract

A hallmark of RNA interference is the production of short double-stranded RNA (dsRNA) molecules 21-28 nucleotides in length by the specialized RNase III protein Dicer. Dicer enzymes uniquely generate RNA products of specific lengths by mechanisms that have not been fully elucidated. Here we show that the PAZ domain responsible for dsRNA end recognition confers this measuring ability through both its structural position and RNA-binding specificity. Point mutations define the dsRNA-binding surface and reveal a protein loop important for cleavage of substrates containing perfect or imperfect base pairing. On the basis of these results, we reengineered Dicer with a U1A RNA-binding domain in place of the PAZ domain to create an enzyme with altered end-recognition specificity and RNA product length. These results explain how Dicer functions as a molecular ruler and provide a structural basis for modifying its activity in cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Giardia lamblia / enzymology
  • Giardia lamblia / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Point Mutation
  • Protein Structure, Tertiary*
  • Protozoan Proteins / chemistry*
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • RNA, Double-Stranded / genetics
  • RNA, Double-Stranded / metabolism*
  • Ribonuclease III / chemistry*
  • Ribonuclease III / genetics
  • Ribonuclease III / metabolism*
  • Static Electricity

Substances

  • Protozoan Proteins
  • RNA, Double-Stranded
  • Ribonuclease III