MicroRNAs align with accessible sites in target mRNAs.
MicroRNAs align with accessible sites in target mRNAs.
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MicroRNA 与目标 mRNA 中的可接近位点对齐。
DOI：
10.1002/jcb.22428
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发表时间：
2010
影响因子：
4
通讯作者：
Clawson,GaryA
中科院分区：
文献类型：
--
作者：
Pan,Weihua;Xin,Ping;Clawson,GaryA
microRNAs（miRs）在基因表达调控中的重要性已被人们清楚地认识到。虽然单个microRNA被认为通过不完全碱基配对靶向数百种不同的mRNA，但对miR靶位点的特征知之甚少。在这里，我们表明miR可以与靶RNA（细胞角蛋白19，KRT）中凭经验鉴定的可接近位点对齐，并且一些对齐的miR在转录后功能上下调KRT表达。我们采用基于RNase-H-的随机文库选择方案来鉴定KRT RNA中的可接近位点。然后，我们将桑格研究所数据库中收集的人类miR与KRT mRNA进行比对，并使用基于网络的MicroInspector程序进行比对。大多数miR与经验鉴定的可接近位点对齐;那些与经验鉴定的位点不对齐的miR在基于RNase-H的测定中也有效地起作用。用第二靶RNA（乳房珠蛋白）获得了类似的结果。瞬时转染测定确定，与KRT对齐的一些miR在蛋白质水平上显著下调KRT，而对RNA水平没有影响。功能有效的miR在KRT的编码区内对齐，而与3′-非翻译区对齐的许多miR不产生下调。J.细胞。109：509-518，2010.© 2009 Wiley利斯公司
The importance of microRNAs (miRs) in control of gene expression is now clearly recognized. While individual microRNAs are thought to target hundreds of disparate mRNAs via imperfect base pairing, little is known about the characteristics of miR target sites. Here we show that the miRs can be aligned with empirically identified accessible sites in a target RNA (Cytokeratin 19, KRT), and that some of the aligned miRs functionally down‐regulate KRT expression post‐transcriptionally. We employed an RNase‐H‐based random library selection protocol to identify accessible sites in KRT RNA. We then aligned the Sanger Institute database collection of human miRs to KRT mRNA, and also aligned them using the web‐based MicroInspector program. Most miRs aligned with the accessible sites identified empirically; those not aligned with the empirically identified sites also functioned effectively in RNase‐H‐based assays. Similar results were obtained with a second target RNA (Mammoglobin). Transient transfection assays established that some of the miRs which aligned with KRT significantly down‐regulated it at the protein level, with no effect on RNA level. The functionally effective miRs aligned within the coding region of KRT, whereas a number of miRs which aligned with the 3′‐untranslated region did not produce down‐regulation. J. Cell. Biochem. 109: 509–518, 2010. © 2009 Wiley‐Liss, Inc.