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, 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.
Institute for Cell Biochemistry and Clinical Neurobiology, University Hospital Hamburg-Eppendorf, D-20246 Hamburg, Germany.
Cornelia
C
Brendel
Monika
M
Rehbein
Hans-Jürgen
HJ
Kreienkamp
Friedrich
F
Buck
Dietmar
D
Richter
Stefan
S
Kindler
D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D046912:Multiprotein Complexes / Q000737:chemistry*; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011132:Polyribosomes / Q000737:chemistry; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D012261:Ribonucleoproteins / Q000737:chemistry; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry; D014162:Transfection / Q000379:methods; D000097796:Nucleolin
D046912:Multiprotein Complexes; D010750:Phosphoproteins; D012313:RNA; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; D000097796:Nucleolin; C492186:Stau1 protein, rat; C054675:ribosomal protein P0