Department of Molecular, Cell and Systems Biology, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521, USA.
Department of Molecular, Cell and Systems Biology, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521, USA.
J Mol Biol. 2018 Feb 2;430(3):285-296. doi: 10.1016/j.jmb.2017.12.006. Epub 2017 Dec 20.
The 3' untranslated region (UTR) of mRNAs is the primary regulatory region that mediates post-transcriptional control by microRNAs and RNA-binding proteins in the cytoplasm. Aside from individual sequence-specific binding and regulation, examples of interaction between these factors at particular 3' UTR sites have emerged. However, the whole picture of such higher-order regulatory modules across the transcriptome is lacking. Here, we investigate the interactions between HuR, a ubiquitous RNA-binding protein, and Ago2, a core effector of the miRNA pathway, at the transcriptome-wide level. Using HITS-CLIP, we map HuR and miRNA binding sites on human 3' UTRs and assess their co-occurrence. In addition, we demonstrate global effects of HuR knockdown on Ago2 occupancy, suggesting a co-regulatory relationship. Focusing on sites of Ago2-HuR overlap, 13 candidates were screened in luciferase reporter assays. Eleven sites showed miRNA-dependent repression, as confirmed in Dicer-null cells. To test for HuR's role in co-regulation, we measured the reporters in HuR KO cells. Three of the miRNA sites demonstrated altered activities, indicating that HuR has an effect on miRNA repression at those sites. Our study presents an efficient search and validation system for studying miRNA-HuR interactions, which expands our understanding of the combinatorial post-transcriptional control of gene expression at the 3' UTR.
mRNA 的 3' 非翻译区（UTR）是主要的调节区域，通过细胞质中的 microRNAs 和 RNA 结合蛋白介导转录后调控。除了个体的序列特异性结合和调节外，这些因素在特定的 3'UTR 位点相互作用的例子也已经出现。然而，整个转录组中这种更高阶的调节模块的全貌还缺乏。在这里，我们在全转录组水平上研究了普遍存在的 RNA 结合蛋白 HuR 和 miRNA 途径的核心效应因子 Ago2 之间的相互作用。我们使用 HITS-CLIP 方法，在人类 3'UTR 上绘制 HuR 和 miRNA 结合位点，并评估它们的共定位。此外，我们还证明了 HuR 敲低对 Ago2 占据的全局影响，表明存在共同调节关系。在 Ago2-HuR 重叠的位点上，我们在荧光素酶报告基因检测中筛选了 13 个候选物。有 11 个位点显示出 miRNA 依赖性抑制，在 Dicer 缺失细胞中得到了证实。为了测试 HuR 在共同调节中的作用，我们在 HuR KO 细胞中测量了报告基因。三个 miRNA 位点显示出活性改变，表明 HuR 对这些位点的 miRNA 抑制有影响。我们的研究提供了一种高效的搜索和验证系统，用于研究 miRNA-HuR 相互作用，扩展了我们对 3'UTR 处基因表达的组合转录后调控的理解。
