Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase

Genes Dev. 2011 Nov 15;25(22):2409-21. doi: 10.1101/gad.175299.111.


Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure-function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1-Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Hydro-Lyases / chemistry
  • Hydro-Lyases / genetics
  • Intramolecular Transferases
  • Microtubule-Associated Proteins / chemistry
  • Microtubule-Associated Proteins / genetics
  • Models, Molecular*
  • Multiprotein Complexes / chemistry
  • Mutation
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • Pseudouridine / metabolism
  • RNA-Binding Proteins / chemistry
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ribonucleoproteins, Small Nuclear / chemistry
  • Ribonucleoproteins, Small Nuclear / genetics
  • Ribonucleoproteins, Small Nuclear / metabolism
  • Ribonucleoproteins, Small Nucleolar / chemistry*
  • Ribonucleoproteins, Small Nucleolar / genetics
  • Ribonucleoproteins, Small Nucleolar / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Alignment


  • Microtubule-Associated Proteins
  • Multiprotein Complexes
  • NOP10 protein, S cerevisiae
  • Nuclear Proteins
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Ribonucleoproteins, Small Nuclear
  • Ribonucleoproteins, Small Nucleolar
  • Saccharomyces cerevisiae Proteins
  • NHP2 protein, S cerevisiae
  • Pseudouridine
  • GAR1 protein, S cerevisiae
  • Hydro-Lyases
  • CBF5 protein, S cerevisiae
  • Intramolecular Transferases
  • pseudouridine synthases