Multimerization of Staufen1 in live cells
Multimerization of Staufen1 in live cells
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DOI：
10.1261/rna.1664210
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发表时间：
2010-03-01
期刊：
影响因子：
4.5
通讯作者：
Desgroseillers, Luc
中科院分区：
文献类型：
--
作者：
Martel, Catherine;Dugre-Brisson, Samuel;Desgroseillers, Luc
信使核糖核酸的转运是将蛋白质靶向细胞特定区域的一种有效机制。虽然已有文献证明，mRNAs是通过核糖核蛋白(RNP)复合体运输的，但涉及复合体形成和定位的几个机制却知之甚少。Staufen(Stau)1是一种双链RNA结合蛋白，是公认的mRNA转运复合体的标志物。在这篇手稿中，我们使用免疫沉淀和生物发光共振能量转移(BRET)分析提供了Stau1在活细胞中自我结合的证据。双链RNA结合域dsRBD3和dsRBD4贡献了大约一半的信号，表明Stau1 RNA结合活性参与了Stau1的自结合。蛋白质之间的相互作用也通过dsRBD5和dsRBD2发生，如体外下拉试验、酵母双杂交和活细胞中的Bret试验所显示的那样。有趣的是，Stau1的自结合有助于低聚复合体的形成，在与Stau1-YFP结合的蛋白质互补试验(PCA)和Bret试验中，与Stau1共价连接的分裂Renilla荧光素酶半部分的共表达证明了这一点。此外，我们还表明，当RNA染色Syto 14作为能量受体进行PCA/Bret分析时，这些含有Stau1的高阶复合体携带RNA。通过两次免疫沉淀和qRT-PCR扩增的生化方法，证实了含有Stau1的复合体的寡聚组成和特定mRNAs的存在。综上所述，这些结果表明，Stau1通过其多个功能结构域在mRNPs中自结合，这些功能结构域可以选择要运输的mRNAs，并建立蛋白质-蛋白质相互作用。
Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.