Isolation of microRNA targets by miRNP immunopurification
Isolation of microRNA targets by miRNP immunopurification
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DOI：
10.1261/rna.563707
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发表时间：
2007-08-01
期刊：
影响因子：
4.5
通讯作者：
Cohen, Stephen M.
中科院分区：
文献类型：
--
作者：
Easow, George;Teleman, Aurelio A.;Cohen, Stephen M.
microRNA（miRNA）通过指导效应子复合物（miRNP）作为基因表达的转录后调节剂，以靶向RNA。尽管在计算方法中取得了很大进展以识别miRNA靶标，但仅报告了对其在体内功能的能力的相对有限的评估。在这里，我们描述了一种基于生化方法的miRNA靶标识别的替代方法，用于纯化具有相关miRNA和绑定mRNA靶标的miRNP复合物。微阵列分析表明，在与miRNP相关的mRNA的3'UTR中，miRNA互补位点的富集高度富集。通过比较缺乏miR-1的野生型蝇和突变果蝇的miRNP相关的mRNA来鉴定与单个miRNA相关的mRNA，并且对miRNA的调节进行了验证。这种方法提供了一种基于体内的物理相互作用来识别功能miRNA靶标的方法。
microRNAs (miRNAs) serve as post-transcriptional regulators of gene expression, by guiding effector complexes (miRNPs) to target RNAs. Although considerable progress has been made in computational methods to identify miRNA targets, only a relatively limited assessment of their ability to function in vivo has been reported. Here we describe an alternative approach to miRNA target identification based on a biochemical method for purifying miRNP complexes with associated miRNAs and bound mRNA targets. Microarray analysis revealed a high degree of enrichment for miRNA complementary sites in the 3'UTRs of the miRNP-associated mRNAs. mRNAs specifically associated with an individual miRNA were identified by comparing the miRNP-associated mRNAs from wild-type flies and mutant flies lacking miR-1, and their regulation by the miRNA was validated. This approach provides a means to identify functional miRNA targets based on their physical interaction in vivo.