Structural basis for 5'-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

Nature. 2005 Mar 31;434(7033):666-70. doi: 10.1038/nature03514.

Abstract

RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold critical for the endonuclease cleavage activity of RISC. Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5'-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5' end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5' end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5' phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere, provide direct support for the 5' region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5' end of the guide RNA.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / metabolism*
  • Archaeoglobus fulgidus / chemistry*
  • Argonaute Proteins
  • Base Sequence
  • Binding Sites
  • Eukaryotic Initiation Factor-2
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Peptide Initiation Factors / chemistry
  • Peptide Initiation Factors / genetics
  • Peptide Initiation Factors / metabolism
  • Phosphates / metabolism
  • Protein Structure, Tertiary
  • RNA Interference
  • RNA, Double-Stranded / chemistry*
  • RNA, Double-Stranded / genetics
  • RNA, Double-Stranded / metabolism*
  • RNA, Guide / chemistry
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • Substrate Specificity

Substances

  • AGO2 protein, human
  • Archaeal Proteins
  • Argonaute Proteins
  • Eukaryotic Initiation Factor-2
  • Peptide Initiation Factors
  • Phosphates
  • RNA, Double-Stranded
  • RNA, Guide
  • RNA, Messenger

Associated data

  • PDB/1YTU