Coller Jeff, Wickens Marv
Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, Ohio, USA.
Methods Enzymol. 2007;429:299-321. doi: 10.1016/S0076-6879(07)29014-7.
Proteins and protein complexes that regulate mRNA metabolism must possess two activities. They bind the mRNA, and then elicit some function, that is, regulate mRNA splicing, transport, localization, translation, or stability. These two activities can often reside in different proteins in a complex, or in different regions of a single polypeptide. Much can be learned about the function of the protein or complex once it is stripped of the constraints imposed by RNA binding. With this in mind, we developed a "tethered function" assay, in which the mRNA regulatory protein is brought to the 3' UTR of an mRNA reporter through a heterologous RNA-protein interaction. In this manner, the functional activity of the protein can be studied independent of its intrinsic ability to recognize and bind to RNA. This simple assay has proven useful in dissecting numerous proteins involved in posttranscriptional regulation. We discuss the basic assay, consider technical issues, and present case studies that exemplify the strengths and limitations of the approach.
调节mRNA代谢的蛋白质和蛋白质复合物必须具备两种活性。它们结合mRNA，然后引发某种功能，即调节mRNA的剪接、转运、定位、翻译或稳定性。这两种活性通常可存在于复合物中的不同蛋白质中，或单一多肽的不同区域。一旦去除RNA结合所带来的限制，就可以了解到很多关于该蛋白质或复合物功能的信息。基于此，我们开发了一种“拴系功能”检测方法，通过异源RNA-蛋白质相互作用将mRNA调节蛋白带到mRNA报告基因的3'UTR。通过这种方式，可以独立于其识别和结合RNA的内在能力来研究该蛋白质的功能活性。这种简单的检测方法已被证明在剖析众多参与转录后调控的蛋白质方面很有用。我们讨论了基本检测方法，考虑了技术问题，并展示了一些案例研究，这些案例体现了该方法的优点和局限性。
