Characterization of Staufen 1 ribonucleoprotein complexes
Characterization of Staufen 1 ribonucleoprotein complexes
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DOI：
10.1042/bj20040812
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发表时间：
2004-12-01
影响因子：
4.1
通讯作者：
Kindler, S
中科院分区：
文献类型：
--
作者：
Brendel, C;Rehbein, M;Kindler, S
在果蝇卵母细胞和神经母细胞中，双链RNA结合蛋白Staufen组装成核糖核蛋白颗粒，介导细胞质mRNA的转运和翻译。两种不同的哺乳动物同源物似乎也存在于不同的含有rna的颗粒中。迄今为止，对含staufen的核糖核蛋白复合物的分子组成知之甚少。在这里，我们使用了一种新的一步亲和纯化方案来鉴定含有Staufen 1的颗粒的成分。核胞质rna结合蛋白核蛋白以rna依赖的方式与Staufen结合，而蛋白磷酸酶1、微管依赖的运动蛋白激酶和一些大小核糖体亚基的组分与Staufen核糖核蛋白复合物的结合是rna独立的。值得注意的是，所有这些成分都不与另一种rna结合蛋白hnRNPK（异质核糖核蛋白K）共纯化，证明了纯化方案的高特异性。此外，拉下和免疫沉淀实验表明，Staufen 1在体外和细胞内与核糖体蛋白PO直接相互作用。在细胞分离和蔗糖梯度实验中，Staufen与完整的核糖体和多体共同分离，但不与分离的40s核糖体亚基共同分离。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募集到含有核糖体的核糖核蛋白颗粒中，该颗粒还含有激酶、蛋白磷酸酶1和核蛋白。
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 PO 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.