The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor

PLoS Genet. 2018 Aug 31;14(8):e1007597. doi: 10.1371/journal.pgen.1007597. eCollection 2018 Aug.

Abstract

The early steps of the production of the large ribosomal subunit are probably the least understood stages of eukaryotic ribosome biogenesis. The first specific precursor to the yeast large ribosomal subunit, the first pre-60S particle, contains 30 assembly factors (AFs), including 8 RNA helicases. These helicases, presumed to drive conformational rearrangements, usually lack substrate specificity in vitro. The mechanisms by which they are targeted to their correct substrate within pre-ribosomal particles and their precise molecular roles remain largely unknown. We demonstrate that the Dbp6p helicase, essential for the normal accumulation of the first pre-60S pre-ribosomal particle in S. cerevisiae, associates with a complex of four AFs, namely Npa1p, Npa2p, Nop8p and Rsa3p, prior to their incorporation into the 90S pre-ribosomal particles. By tandem affinity purifications using yeast extracts depleted of one component of the complex, we show that Npa1p forms the backbone of the complex. We provide evidence that Npa1p and Npa2p directly bind Dbp6p and we demonstrate that Npa1p is essential for the insertion of the Dbp6p helicase within 90S pre-ribosomal particles. In addition, by an in vivo cross-linking analysis (CRAC), we map Npa1p rRNA binding sites on 25S rRNA adjacent to the root helices of the first and last secondary structure domains of 25S rRNA. This finding supports the notion that Npa1p and Dbp6p function in the formation and/or clustering of root helices of large subunit rRNAs which creates the core of the large ribosomal subunit RNA structure. Npa1p also crosslinks to snoRNAs involved in decoding center and peptidyl transferase center modifications and in the immediate vicinity of the binding sites of these snoRNAs on 25S rRNA. Our data suggest that the Dbp6p helicase and the Npa1p complex play key roles in the compaction of the central core of 25S rRNA and the control of snoRNA-pre-rRNA interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DEAD-box RNA Helicases / metabolism
  • Escherichia coli
  • Models, Molecular
  • Molecular Chaperones / metabolism*
  • Nuclear Proteins / metabolism*
  • Peptidyl Transferases / metabolism
  • Protein Interaction Domains and Motifs
  • Protein Structure, Secondary
  • RNA Helicases / metabolism*
  • RNA Precursors / metabolism
  • RNA, Ribosomal / metabolism
  • RNA, Small Nucleolar / metabolism
  • RNA-Binding Proteins / metabolism
  • Recombinant Proteins
  • Ribosomal Proteins / metabolism
  • Ribosome Subunits, Large, Eukaryotic / metabolism*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Substrate Specificity
  • Trans-Activators / metabolism

Substances

  • Molecular Chaperones
  • Nop8 protein, S cerevisiae
  • Nuclear Proteins
  • RNA Precursors
  • RNA, Ribosomal
  • RNA, Small Nucleolar
  • RNA-Binding Proteins
  • RSA3 protein, S cerevisiae
  • Recombinant Proteins
  • Ribosomal Proteins
  • Saccharomyces cerevisiae Proteins
  • Trans-Activators
  • URB1 protein, S cerevisiae
  • URB2 protein, S cerevisiae
  • RNA, ribosomal, 25S
  • Peptidyl Transferases
  • DBP6 protein, S cerevisiae
  • DEAD-box RNA Helicases
  • RNA Helicases

Grants and funding

French Ministry of Research PhD fellowship and Paul Sabatier University ATER contract [to CJ]; Centre National de la Recherche Scientifique (CNRS); The University of Toulouse; and Agence Nationale de la Recherche (ANR, http://www.agence-nationale-recherche.fr/) [ANR-2010-BLAN-1224 to YH]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.