MS2-TRAP (MS2-tagged RNA affinity purification): tagging RNA to identify associated miRNAs.
MS2-TRAP (MS2-tagged RNA affinity purification): tagging RNA to identify associated miRNAs.
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DOI：
10.1016/j.ymeth.2012.07.004
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发表时间：
2012-10
期刊：
影响因子：
4.8
通讯作者：
Gorospe, Myriam
中科院分区：
文献类型：
--
作者：
Yoon, Je-Hyun;Srikantan, Subramanya;Gorospe, Myriam
关键词：
所有类型的细胞转录产物，包括编码信使(M)RNAsx和非编码(NC)RNA，都受到广泛的转录后调控。在引起转录后调控的因素中，microRNAs(MiRNAs)已经成为一类主要的小调控RNA。由于RNA-RNA相互作用可以通过计算进行建模，因此已经开发了几个优秀的程序来预测miRNAs与目标转录本的相互作用。然而，由于不同的原因，许多这样的预测没有实现，包括细胞中miRNA的缺失或低丰度表达，竞争因子的存在或掩盖了microRNA位点的构象变化，以及目标转录产物不存在于预测数据库中的可能性，就像长的ncRNAs一样。在这里，我们提供了一种称为MS2-TRAP(标记RNA亲和纯化)的系统方法，用于在细胞环境中识别与目标转录本相关的miRNAs。我们根据MS2 RNA发夹标记的lincRNA(lincRNA-p21-MS2)的表达和识别MS2 RNA发夹的融合蛋白MS2-GST的伴随表达，通过识别与长基因间(Li)ncRNA相关的microRNA来说明这一方法的使用。亲和下拉由[MS2-GST/lincRNA-p21-MS2]组成的核糖核蛋白(RNP)复合体后，分离下拉材料中的RNA并进行逆转录(RT)。随后通过使用实时定量(Q)PCR分析对存在于下拉复合体中的microRNAs进行评估，从而鉴定出与lincRNA-p21相互作用并控制其丰度的真正miRNAs。我们描述了这种方法的替代设计和应用，并讨论了它在破译转录后基因调控方案中的意义。
Cellular transcripts of all types, including coding messenger (m)RNAsx and noncoding (nc)RNAs, are subject to extensive post-transcriptional regulation. Among the factors that elicit post-transcriptional control, microRNAs (miRNAs) have emerged as a major class of small regulatory RNAs. Since RNA-RNA interactions can be modeled computationally, several excellent programs have been developed to predict the interaction of miRNAs with target transcripts. However, many such predictions are not realized for different reasons, including absent or low-abundance expression of the miRNA in the cell, the existence of competing factors or conformational changes masking the microRNA site, and the possibility that target transcripts are not present in the prediction databases, as is the case for long ncRNAs. Here, we provide a systematic approach termed MS2-TRAP (tagged RNA affinity purification) for identifying miRNAs associated with a target transcript in the cellular context. We illustrate the use of this methodology by identifying microRNAs that associate with a long intergenic (li)ncRNA, based on the expression of the lincRNA tagged with MS2 RNA hairpins (lincRNA-p21-MS2) and the concomitant expression of a fusion protein recognizing the MS2 RNA hairpins, MS2-GST. After affinity pulldown of the ribonucleoprotein (RNP) complex comprising [MS2-GST / lincRNA-p21-MS2], the RNA in the pulldown material was isolated and reverse transcribed (RT). Subsequent assessment of the microRNAs present in the pulldown complex by using real-time quantitative (q)PCR analysis led to the identification of bona fide miRNAs that interact with and control the abundance of lincRNA-p21. We describe alternative designs and applications of this approach, and discuss its implications in deciphering post-transcriptional gene regulatory schemes.
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