miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity.
miRNA-target chimeras reveal miRNA 3'-end pairing as a major determinant of Argonaute target specificity.
复制标题
miRNA-target嵌合体揭示了miRNA 3'-end配对，这是Argonaute目标特异性的主要决定因素。
DOI：
10.1038/ncomms9864
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发表时间：
2015-11-25
影响因子：
16.6
通讯作者：
Darnell RB
中科院分区：
文献类型：
--
作者：
Moore MJ;Scheel TK;Luna JM;Park CY;Fak JJ;Nishiuchi E;Rice CM;Darnell RB
微小RNA（miRNAs）作为Argonaute（AGO）蛋白的序列特异性向导，其介导靶信使RNA的转录后沉默。尽管它们在许多生物过程中很重要，但AGO-miRNA靶向的规则仅被部分理解。在这里，我们报告了一种改良的AGO HITS-CLIP策略，称为CLEAR（内源Argonaute结合RNA的共价连接）-CLIP，它富集了连接到其内源mRNA靶点的miRNA。CLEAR-CLIP在小鼠脑中绘制了约130，000个内源性miRNA-靶标相互作用，在人类肝癌细胞中绘制了约40，000个内源性miRNA-靶标相互作用。基序和结构分析定义了超过200种哺乳动物miRNA的扩展配对规则。大多数相互作用结合了联合收割机基于种子的配对与不同的、miRNA特异性的辅助配对模式。在某些调控位点，这种特异性赋予具有共享种子序列但3′端不同的miRNA家族成员不同的沉默功能。这项工作提供了一种明确的生物化学鉴定体内miRNA位点的方法，导致发现miRNA 3′端配对是AGO结合特异性的一般决定因素。 microRNA（miRNAs）充当Argonaute（AGO）蛋白的序列特异性向导。通过使用一种改良的AGO HITS-CLIP策略，该策略富集了与其内源性mRNA靶点连接的miRNA，在这里，作者表明miRNA 3'末端配对是AGO结合特异性的一般决定因素。
microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO–miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA–target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3′-ends. This work provides a means for explicit biochemical identification of miRNA sites in vivo, leading to the discovery that miRNA 3′-end pairing is a general determinant of AGO binding specificity. microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins. By using a modified AGO HITS-CLIP strategy that enriches miRNAs ligated to their endogenous mRNA targets, here the authors show that miRNA 3' end pairing is a general determinant of AGO binding specificity.