Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

J Cell Biol. 2014 Nov 24;207(4):463-80. doi: 10.1083/jcb.201404160. Epub 2014 Nov 17.

Abstract

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATPases Associated with Diverse Cellular Activities
  • Amino Acid Sequence
  • Binding Sites
  • Carrier Proteins / metabolism
  • Cell Line, Tumor
  • Crystallography, X-Ray
  • DNA Helicases / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • HEK293 Cells
  • HSP90 Heat-Shock Proteins / metabolism
  • HeLa Cells
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Kruppel-Like Factor 6
  • Kruppel-Like Transcription Factors / metabolism
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / metabolism
  • Protein Binding
  • Proteomics / methods
  • Proto-Oncogene Proteins / metabolism
  • RNA-Binding Proteins / metabolism
  • Ribonucleases / metabolism
  • Ribonucleoproteins, Small Nuclear / chemistry*
  • Ribonucleoproteins, Small Nuclear / metabolism
  • Ribonucleoproteins, Small Nucleolar / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Sequence Alignment
  • Transcription Factors

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • HSP90 Heat-Shock Proteins
  • KLF6 protein, human
  • Kruppel-Like Factor 6
  • Kruppel-Like Transcription Factors
  • NUFIP1 protein, human
  • NUFIP2 protein, human
  • Neoplasm Proteins
  • Nuclear Proteins
  • Proto-Oncogene Proteins
  • RNA-Binding Proteins
  • Ribonucleoproteins, Small Nuclear
  • Ribonucleoproteins, Small Nucleolar
  • Snu13 protein, human
  • Transcription Factors
  • ZNF331 protein, human
  • ZNHIT3 protein, human
  • Ribonucleases
  • ATPases Associated with Diverse Cellular Activities
  • DNA Helicases
  • RUVBL1 protein, human
  • RUVBL2 protein, human

Associated data

  • PDB/1ZWZ
  • PDB/3NMU
  • PDB/3PLA