由抑制性微RNA(miRNA)诱导的基因沉默在调控基因表达中的重要性现已非常明显.但因为一个信使RNA(mRNA)的miRNA调控在它们的序列中只需要有一个短的匹配段(长度为8个核苷酸或更少),所以事实证明,要明确确定所预测的很多mRNA结合点中哪个是每个miRNA在活体中的目标几乎不可能. 现在,对HITS-CLIP方法所做的一种改进(这种改进的方法以miRNA和 mRNA与Argonaute蛋白的相互作用为关注点;Argonaute蛋白是普遍存在的核酸内切酶,它们形成由RNA诱导的沉默复合物的一部分),被用来解析miRNA与大脑mRNA转录体的结合点的一个精确分布图.该方法具有普遍适用性,应能为了解miRNA在生物学中的作用提供了一个新手段. 另外,这种分布图可让研究人员确定具有临床意义的mRNA上用于RNA干涉(RNAi)疗法的目标作用点.

    Argonaute HITS-CLIP decodes microRNA–mRNA interaction maps Sung Wook Chi1, Julie B. Zang1, Aldo Mele1 & Robert B. Darnell1 1 Laboratory of Molecular Neuro-Oncology and Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA MicroRNAs (miRNAs) have critical roles in the regulation of gene expression; however, as miRNA activity requires base pairing with only 6-8 nucleotides of messenger RNA, predicting target mRNAs is a major challenge. Recently, high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) has identified functional protein–RNA interaction sites. Here we use HITS-CLIP to covalently crosslink native argonaute (Ago, also called Eif2c) protein–RNA complexes in mouse brain. This produced two simultaneous data sets—Ago–miRNA and Ago–mRNA binding sites—that were combined with bioinformatic analysis to identify interaction sites between miRNA and target mRNA. We validated genome-wide interaction maps for miR-124, and generated additional maps for the 20 most abundant miRNAs present in P13 mouse brain. Ago HITS-CLIP provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA–mRNA interactions.

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