Characterization of Staufen 1 ribonucleoprotein complexes

• Published in: Biochemical journal Vol. 384; no. Pt 2; pp. 239 - 246
• Main Authors: Brendel, Cornelia, Rehbein, Monika, Kreienkamp, Hans-Jürgen, Buck, Friedrich, Richter, Dietmar, Kindler, Stefan
• Format: Journal Article
• Language: English
• Published: England Portland Press Ltd 01.12.2004
• Subjects: Animals; Brain - cytology; Brain - metabolism; Cell Fractionation - methods; Cells, Cultured; Chromatography, Affinity - methods; Drosophila; Humans; Kidney - chemistry; Kidney - cytology; Kidney - embryology; Kidney - metabolism; Multiprotein Complexes - chemistry; Neurons - chemistry; Neurons - metabolism; Nucleolin; Phosphoproteins - metabolism; Polyribosomes - chemistry; Rats; Ribonucleoproteins - chemistry; Ribosomal Proteins - metabolism; Ribosomes - chemistry; RNA - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Transfection - methods
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20040812

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.