种子序列之外的配对支持微小RNA靶向特异性。
Pairing beyond the Seed Supports MicroRNA Targeting Specificity.
作者信息
Broughton James P, Lovci Michael T, Huang Jessica L, Yeo Gene W, Pasquinelli Amy E
机构信息
Division of Biology, University of California, San Diego, La Jolla, CA 92093-0349, USA.
Department of Cellular and Molecular Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA.
出版信息
Mol Cell. 2016 Oct 20;64(2):320-333. doi: 10.1016/j.molcel.2016.09.004. Epub 2016 Oct 6.
To identify endogenous miRNA-target sites, we isolated AGO-bound RNAs from Caenorhabditis elegans by individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP), which fortuitously also produced miRNA-target chimeric reads. Through the analysis of thousands of reproducible chimeras, pairing to the miRNA seed emerged as the predominant motif associated with functional interactions. Unexpectedly, we discovered that additional pairing to 3' sequences is prevalent in the majority of target sites and leads to specific targeting by members of miRNA families. By editing an endogenous target site, we demonstrate that 3' pairing determines targeting by specific miRNA family members and that seed pairing is not always sufficient for functional target interactions. Finally, we present a simplified method, chimera PCR (ChimP), for the detection of specific miRNA-target interactions. Overall, our analysis revealed that sequences in the 5' as well as the 3' regions of a miRNA provide the information necessary for stable and specific miRNA-target interactions in vivo.
为了鉴定内源性miRNA靶位点，我们通过单核苷酸分辨率交联免疫沉淀（iCLIP）从秀丽隐杆线虫中分离AGO结合的RNA，该方法偶然也产生了miRNA-靶嵌合读数。通过对数千个可重复嵌合体的分析，与miRNA种子配对成为与功能相互作用相关的主要基序。出乎意料的是，我们发现大多数靶位点中与3'序列的额外配对很普遍，并导致miRNA家族成员的特异性靶向。通过编辑内源性靶位点，我们证明3'配对决定了特定miRNA家族成员的靶向，并且种子配对对于功能性靶相互作用并不总是足够的。最后，我们提出了一种简化的方法，嵌合体PCR（ChimP），用于检测特定的miRNA-靶相互作用。总体而言，我们的分析表明，miRNA的5'和3'区域中的序列为体内稳定和特异性的miRNA-靶相互作用提供了必要的信息。
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