Yoon Je-Hyun, Gorospe Myriam
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA.
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Laboratory of Genetics and Genomics, National Institute on Aging-Intramural Research Program, National Institutes of Health, 173 Ashley Avenue, Charleston, SC, 29425, USA.
Methods Mol Biol. 2016;1421:15-22. doi: 10.1007/978-1-4939-3591-8_2.
Posttranscriptional gene expression is governed by the interaction of mRNAs with vast families of RNA-binding proteins (RBPs) and noncoding (nc)RNAs. RBPs and ncRNAs jointly influence all aspects of posttranscriptional metabolism, including pre-mRNA splicing and maturation, mRNA transport, editing, stability, and translation. Given the impact of mRNA-interacting molecules on gene expression, there is great interest in identifying mRNA-binding factors comprehensively. Here, we provide a detailed protocol to tag mRNAs with MS2 hairpins and then affinity-purify trans-binding factors (RBPs, ncRNAs) associated with the MS2-tagged mRNA. This method, termed MS2-TRAP, permits the systematic characterization of ribonucleoprotein (RNP) complexes formed on a given mRNA of interest. We describe how to prepare the mRNA-MS2 expression vector, purify the MS2-tagged RNP complexes, and detect bound RNAs and RBPs, as well as variations of this methodology to address related questions of RNP biology.
转录后基因表达受信使核糖核酸（mRNA）与大量核糖核酸结合蛋白（RBP）家族及非编码（nc）RNA相互作用的调控。RBP和ncRNA共同影响转录后代谢的各个方面，包括前体mRNA剪接与成熟、mRNA转运、编辑、稳定性及翻译。鉴于与mRNA相互作用的分子对基因表达的影响，人们对全面鉴定mRNA结合因子有着浓厚兴趣。在此，我们提供了一个详细方案，用MS2发夹标记mRNA，然后亲和纯化与MS2标记的mRNA相关的反式结合因子（RBP、ncRNA）。这种方法称为MS2-TRAP，可对在给定的目标mRNA上形成的核糖核蛋白（RNP）复合物进行系统表征。我们描述了如何制备mRNA-MS2表达载体、纯化MS2标记的RNP复合物，以及检测结合的RNA和RBP，还有该方法的变体以解决RNP生物学的相关问题。
