Characterization of Staufen 1 ribonucleoprotein complexes

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, rela...

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Veröffentlicht in:Biochemical journal 2004-12, Vol.384 (Pt 2), p.239-246
Hauptverfasser: Brendel, Cornelia, Rehbein, Monika, Kreienkamp, Hans-Jürgen, Buck, Friedrich, Richter, Dietmar, Kindler, Stefan
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container_end_page 246
container_issue Pt 2
container_start_page 239
container_title Biochemical journal
container_volume 384
creator Brendel, Cornelia
Rehbein, Monika
Kreienkamp, Hans-Jürgen
Buck, Friedrich
Richter, Dietmar
Kindler, Stefan
description 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.
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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. 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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
title Characterization of Staufen 1 ribonucleoprotein complexes
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