Identification of human microRNA targets from isolated argonaute protein complexes
Identification of human microRNA targets from isolated argonaute protein complexes
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DOI：
10.4161/rna.4.2.4640
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发表时间：
2007-04-01
期刊：
影响因子：
4.1
通讯作者：
Meister, Gunter
中科院分区：
文献类型：
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作者：
Beitzinger, Michaela;Peters, Lasse;Meister, Gunter
MicroRNA（miRNAs）是一类在翻译和/或mRNA稳定性水平上调控基因表达的小分子非编码RNA。哺乳动物miRNA与Argonaute（Ago）蛋白家族的成员结合，并与特定靶mRNA的3'非翻译区（UTR）中的部分互补序列结合。基于自由结合能或序列保守性等因素的计算机算法已被用于预测miRNA靶mRNA。基于这样的预测，多达三分之一的哺乳动物mRNA似乎处于miRNA调控之下。然而，由于miRNA与其靶标之间的互补性程度低，这样的计算机程序通常不精确，因此不是非常可靠。在这里，我们报告的第一个生物化学鉴定方法的miRNA目标从人类细胞。使用高特异性的单克隆抗体对Ago蛋白家族的成员，我们共免疫沉淀Ago结合的mRNA，并通过克隆来识别它们。有趣的是，大多数已确定的目标也是由不同的计算机程序预测的。此外，我们随机分析了六种不同的候选靶点，并能够通过实验验证五种作为miRNA靶点。我们的数据清楚地表明，miRNA的目标可以通过实验从Ago复合物中鉴定出来，因此提供了一种直接分析miRNA功能的新工具。
MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, we co-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function.