Characterization of Staufen 1 ribonucleoprotein complexes

Biochem J. 2004 Dec 1;384(Pt 2):239-46. doi: 10.1042/BJ20040812.

Abstract

In Drosophila oocytes and neuroblasts, the double-stranded RNA binding protein Staufen assembles into ribonucleoprotein particles, which mediate cytoplasmic mRNA trafficking and translation. Two different mammalian orthologues also appear to reside in distinct RNA-containing particles. To date, relatively little is known about the molecular composition of Staufen-containing ribonucleoprotein complexes. Here, we have used a novel one-step affinity purification protocol to identify components of Staufen 1-containing particles. Whereas the nucleocytoplasmic RNA-binding protein nucleolin is linked to Staufen in an RNA-dependent manner, the association of protein phosphatase 1, the microtubule-dependent motor protein kinesin and several components of the large and small ribosomal subunits with Staufen ribonucleoprotein complexes is RNA-independent. Notably, all these components do not co-purify with a second RNA-binding protein, hnRNPK (heterogeneous ribonucleoprotein K), demonstrating the high specificity of the purification protocol. Furthermore, pull-down and immunoprecipitation experiments suggest a direct interaction between Staufen 1 and the ribosomal protein P0 in vitro as well as in cells. In cell fractionation and sucrose gradient assays, Staufen co-fractionates with intact ribosomes and polysomes, but not with the isolated 40 S ribosomal subunit. Taken together, these findings imply that, in the cytoplasm of mammalian cells, an association with the ribosomal P-stalk protein P0 recruits Staufen 1 into ribosome-containing ribonucleoprotein particles, which also contain kinesin, protein phosphatase 1 and nucleolin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / cytology
  • Brain / metabolism
  • Cell Fractionation / methods
  • Cells, Cultured
  • Chromatography, Affinity / methods
  • Humans
  • Kidney / chemistry
  • Kidney / cytology
  • Kidney / embryology
  • Kidney / metabolism
  • Multiprotein Complexes / chemistry*
  • Neurons / chemistry
  • Neurons / metabolism
  • Phosphoproteins / metabolism
  • Polyribosomes / chemistry
  • RNA / metabolism
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / metabolism
  • Rats
  • Ribonucleoproteins / chemistry
  • Ribosomal Proteins / metabolism
  • Ribosomes / chemistry
  • Transfection / methods

Substances

  • Multiprotein Complexes
  • Phosphoproteins
  • RNA-Binding Proteins
  • Ribonucleoproteins
  • Ribosomal Proteins
  • Stau1 protein, rat
  • nucleolin
  • ribosomal protein P0
  • RNA