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Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface.

Journal of molecular biology | 2011

H/ACA small nucleolar and Cajal body ribonucleoproteins (RNPs) function in site-specific pseudouridylation of eukaryotic rRNA and snRNA, rRNA processing, and vertebrate telomerase biogenesis. Nhp2, one of four essential protein components of eukaryotic H/ACA RNPs, forms a core trimer with the pseudouridylase Cbf5 and Nop10 that binds to H/ACA RNAs specifically. Crystal structures of archaeal H/ACA RNPs have revealed how the protein components interact with each other and with the H/ACA RNA. However, in place of Nhp2p, archaeal H/ACA RNPs contain L7Ae, which binds specifically to an RNA K-loop motif absent from eukaryotic H/ACA RNPs, while Nhp2 binds a broader range of RNA structures. We report solution NMR studies of Saccharomyces cerevisiae Nhp2 (Nhp2p), which reveal that Nhp2p exhibits two major conformations in solution due to cis/trans isomerization of the evolutionarily conserved Pro83. The equivalent proline is in the cis conformation in all reported structures of L7Ae and other homologous proteins. Nhp2p has the expected α-β-α fold, but the solution structures of the major conformation of Nhp2p with trans Pro83 and of Nhp2p-S82W with cis Pro83 reveal that Pro83 cis/trans isomerization affects the positions of numerous residues at the Nop10 and RNA binding interface. An S82W substitution, which stabilizes the cis conformation, also stabilizes the association of Nhp2p with H/ACA snoRNPs expressed in vivo. We propose that Pro83 plays a key role in the assembly of the eukaryotic H/ACA RNP, with the cis conformation locking in a stable Cbf5-Nop10-Nhp2 ternary complex and positioning the protein backbone to interact with the H/ACA RNA.

Pubmed ID: 21708174 RIS Download

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Associated grants

  • Agency: NIGMS NIH HHS, United States
    Id: R01 GM037254
  • Agency: NIGMS NIH HHS, United States
    Id: R01 GM048123
  • Agency: NIGMS NIH HHS, United States
    Id: GM37254
  • Agency: NIGMS NIH HHS, United States
    Id: GM61518
  • Agency: NIGMS NIH HHS, United States
    Id: R01 GM037254-24
  • Agency: NIGMS NIH HHS, United States
    Id: R01 GM048123-19
  • Agency: NIGMS NIH HHS, United States
    Id: GM48123
  • Agency: NIGMS NIH HHS, United States
    Id: R01 GM061518

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SGD (tool)

RRID:SCR_004694

A curated database that provides comprehensive integrated biological information for Saccharomyces cerevisiae along with search and analysis tools to explore these data. SGD allows researchers to discover functional relationships between sequence and gene products in fungi and higher organisms. The SGD also maintains the S. cerevisiae Gene Name Registry, a complete list of all gene names used in S. cerevisiae which includes a set of general guidelines to gene naming. Protein Page provides basic protein information calculated from the predicted sequence and contains links to a variety of secondary structure and tertiary structure resources. Yeast Biochemical Pathways allows users to view and search for biochemical reactions and pathways that occur in S. cerevisiae as well as map expression data onto the biochemical pathways. Literature citations are provided where available.

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CYANA (tool)

RRID:SCR_014229

Software for automated structure calculation of biological macromolecules on basis of conformational constraints from nuclear magnetic resonance. Program for automated NMR protein structure calculation. CYANA requires a sufficient list of assigned chemical shifts and lists of cross-peak positions and columns from 2D, 3D, or4D NOESY spectra in order to calculate the assignment of the NOESY cross-peaks and the 3D structure of the protein in solution.

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