Isolation and characterization of Staufen-containing ribonucleoprotein particles from rat brain
Isolation and characterization of Staufen-containing ribonucleoprotein particles from rat brain
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DOI：
10.1073/pnas.0334355100
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发表时间：
2003-02-18
影响因子：
11.1
通讯作者：
Kiebler, MA
中科院分区：
文献类型：
--
作者：
Mallardo, M;Deitinghoff, A;Kiebler, MA
局部化的mRNA被认为是以定义的颗粒运输到它们的最终目的地。这些颗粒代表可能参与识别、传输和锚定局部消息的大型蛋白质复合体。然而，这些核核微粒的成分还很少被确定。我们选择了对参与RNA运输的天然RNA-蛋白质复合体进行生化富集化的策略，以确定相关的RNA和蛋白质。由于Staufen蛋白与细胞内RNA转运有关，因此我们选择哺乳动物Staufen蛋白作为纯化RNA转运颗粒的标志物。在这里，我们提出了Staufen蛋白存在于两个不同的复合体中的证据：(I)明显的大的、含有核糖体和内质网的颗粒，在差速离心法中优先在膜颗粒中发现，以及(它)来自大鼠脑匀浆的S100中的较小颗粒。在S100的凝胶过滤中，我们鉴定出含有670 kDa Staufen1和440 kDa Staufen2的可溶性颗粒。它们不与核糖体和内质网共分，而是与动蛋白重链共富。此外，含有Staufen1颗粒的组份显示，与对照组份相比，mRNAs的丰度是对照组的15倍。最重要的是，这些组分高度富含Bc1，在较小程度上，在钙/钙调蛋白依赖激酶II的阿尔法亚单位中，有两个树突定位的RNA。最后，这两种RNA都与Staufen1血凝素共存于转基因海马神经元树突中的颗粒中。因此，我们认为这些含有Staufen1的颗粒可能代表着在神经元内运输到其最终目的地的RNA转运中间体。
Localized mRNAs are thought to be transported in defined particles to their final destination. These particles represent large protein complexes that may be involved in recognizing, transporting, and anchoring localized messages. Few components of these ribo-nucleoparticles, however, have been identified yet. We chose the strategy to biochemically enrich native RNA-protein complexes involved in RNA transport to identify the associated RNAs and proteins. Because Staufen proteins were implicated in intracellular RNA transport, we chose mammalian Staufen proteins as markers for the purification of RNA transport particles. Here, we present evidence that Staufen proteins exist in two different complexes: (i) distinct large, ribosome- and endoplasmic reticulum-containing granules preferentially found in the membrane pellets during differential centrifugation and (it) smaller particles in the S100 from rat brain homogenates. On gel filtration of the S100, we identified soluble 670-kDa Staufen1-containing and 440-kDa Staufen2-containing particles. They do not cofractionate with ribosomes and endoplasmic reticulum but rather coenrich with kinesin heavy chain. Furthermore, the fractions containing the Staufen1 particles show a 15-fold enrichment of mRNAs compared with control fractions. Most importantly, these fractions are highly enriched in BC1, and, to a lesser extent, in the alpha-subunit of the Ca2+/calmodulin-dependent kinase II, two dendritically localized RNAs. Finally, both RNAs colocalize with Staufen1-hemagglutinin in particles in dendrites of transfected hippocampal neurons. We therefore propose that these Staufen1-containing particles may represent RNA transport intermediates that are in transit to their final destination within neurons.