Brendel Cornelia, Rehbein Monika, Kreienkamp Hans-Jürgen, Buck Friedrich, Richter Dietmar, Kindler Stefan
Institute for Cell Biochemistry and Clinical Neurobiology, University Hospital Hamburg-Eppendorf, D-20246 Hamburg, Germany.
Biochem J. 2004 Dec 1;384(Pt 2):239-46. 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.
在果蝇卵母细胞和成神经细胞中,双链RNA结合蛋白Staufen组装成核糖核蛋白颗粒,介导细胞质mRNA的运输和翻译。两种不同的哺乳动物直系同源物似乎也存在于不同的含RNA颗粒中。迄今为止,关于含Staufen的核糖核蛋白复合物的分子组成了解相对较少。在这里,我们使用了一种新颖的一步亲和纯化方案来鉴定含Staufen 1颗粒的成分。核质RNA结合蛋白核仁素以RNA依赖的方式与Staufen相连,而蛋白磷酸酶1、微管依赖的运动蛋白驱动蛋白以及大小核糖体亚基的几个成分与Staufen核糖核蛋白复合物的结合是RNA非依赖的。值得注意的是,所有这些成分都不会与另一种RNA结合蛋白hnRNPK(不均一核糖核蛋白K)共同纯化,这证明了纯化方案的高特异性。此外,下拉和免疫沉淀实验表明,Staufen 1与核糖体蛋白P0在体外以及在细胞中存在直接相互作用。在细胞分级分离和蔗糖梯度分析中,Staufen与完整核糖体和多核糖体共分级,但不与分离的40S核糖体亚基共分级。综上所述,这些发现表明,在哺乳动物细胞的细胞质中,与核糖体P柄蛋白P0的结合将Staufen 1招募到含核糖体的核糖核蛋白颗粒中,这些颗粒还含有驱动蛋白、蛋白磷酸酶1和核仁素。