Mutational Analysis of the Nsa2 N-Terminus Reveals Its Essential Role in Ribosomal 60S Subunit Assembly

Int J Mol Sci. 2020 Nov 30;21(23):9108. doi: 10.3390/ijms21239108.


The ribosome assembly factor Nsa2 is part of the Rea1-Rsa4-Nsa2 interconnected relay on nuclear pre-60S particles that is essential for 60S ribosome biogenesis. Cryo-EM structures depict Nsa2 docked via its C-terminal β-barrel domain to nuclear pre-60S particles, whereas the extended N-terminus, consisting of three α-helical segments, meanders between various 25S rRNA helices with the extreme N-terminus in close vicinity to the Nog1 GTPase center. Here, we tested whether this unappreciated proximity between Nsa2 and Nog1 is of functional importance. Our findings demonstrate that a conservative mutation, Nsa2 Q3N, abolished cell growth and impaired 60S biogenesis. Subsequent genetic and biochemical analyses verified that the Nsa2 N-terminus is required to target Nsa2 to early pre-60S particles. However, overexpression of the Nsa2 N-terminus abolished cytoplasmic recycling of the Nog1 GTPase, and both Nog1 and the Nsa2-N (1-58) construct, but not the respective Nsa2-N (1-58) Q3N mutant, were found arrested on late cytoplasmic pre-60S particles. These findings point to specific roles of the different Nsa2 domains for 60S ribosome biogenesis.

Keywords: Mdn1; Nog1; Nsa2; Rea1; Rsa4; TINP1; ribosome assembly; ribosome biogenesis.

MeSH terms

  • Amino Acid Sequence
  • Catalytic Domain
  • DNA Mutational Analysis
  • GTP-Binding Proteins / metabolism
  • Models, Molecular
  • Mutant Proteins / chemistry
  • Mutant Proteins / metabolism
  • Mutation / genetics
  • Nuclear Proteins / metabolism
  • Phenotype
  • Ribosomal Proteins / chemistry*
  • Ribosomal Proteins / genetics*
  • Ribosomal Proteins / metabolism
  • Ribosome Subunits, Large, Eukaryotic / metabolism*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism


  • Mutant Proteins
  • NOG1 protein, S cerevisiae
  • Nsa2 protein, S cerevisiae
  • Nuclear Proteins
  • Ribosomal Proteins
  • Saccharomyces cerevisiae Proteins
  • GTP-Binding Proteins