Translational control assessed using the tethered function assay in Xenopus oocytes.
Translational control assessed using the tethered function assay in Xenopus oocytes.
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DOI：
10.1016/j.ymeth.2010.02.018
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发表时间：
2010-05
期刊：
影响因子：
4.8
通讯作者：
N. Minshall;Rachel Allison;A. Marnef;A. Wilczyńska;N. Standart
中科院分区：
文献类型：
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作者：
N. Minshall;Rachel Allison;A. Marnef;A. Wilczyńska;N. Standart
束缚功能测定是一种旨在解决RNA结合蛋白在报告RNA代谢中的作用的方法。该测定的基础是通过使用无关的噬菌体MS 2或λ N RNA-蛋白质相互作用来人工地将测试蛋白质拴系到报告mRNA，并评估测试蛋白质对报告RNA的影响。在这一章中，我们首先讨论的原则和有效性的拴系功能的方法，从几种细胞类型和许多蛋白质，调节RNA在各种过程中，包括RNA加工（剪接，聚腺苷酸化/deadenylation，衰变），本地化和蛋白质合成适当的例子。其次，我们将专注于使用这种方法来监测非洲爪蟾卵母细胞中的翻译激活和抑制，给出了详细的协议，并讨论了可能的优化，我们已经探索。
The tethered function assay is a method designed to address the role of an RNA-binding protein upon the metabolism of a reporter RNA. The basis of this assay is to artificially tether a test protein to a reporter mRNA by employing an unrelated bacteriophage MS2 or lambda N RNA–protein interaction, and to assess the effects of the test protein on the reporter RNA. In this chapter, we first discuss the principles and validity of the tethered function approach, drawing on appropriate examples from several cell types and of many proteins that regulate RNA in a variety of processes, including RNA processing (splicing, polyadenylation/deadenylation, decay), localisation and protein synthesis. Secondly, we will focus on the use of this approach to monitor translational activation and repression in Xenopus oocytes, giving a detailed protocol, and discussing possible optimizations we have explored.