Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution.
Resolving Subcellular miRNA Trafficking and Turnover at Single-Molecule Resolution.
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DOI：
10.1016/j.celrep.2017.03.075
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发表时间：
2017-04-18
期刊：
影响因子：
8.8
通讯作者：
Walter NG
中科院分区：
文献类型：
--
作者：
Pitchiaya S;Heinicke LA;Park JI;Cameron EL;Walter NG
miRNA定位和稳定性的调节对于它们广泛的细胞质RNA沉默活性和新兴的核功能至关重要。在这里，我们开发了基于单分子荧光的工具来评估miRNA的亚细胞运输，完整性和活性。我们发现，种子匹配的RNA靶标保护miRNA免受降解，并增强其核保留。虽然靶稳定的功能性细胞质miRNA存在于高分子量复合物中，但核miRNA以及互补抗miRNA靶向的细胞质miRNA稳定地隔离在显著较低分子量的复合物内并使其不能抑制。miRNA的稳定性和活性取决于Argonaute蛋白的丰度，而miRNA链的选择、解旋和核保留取决于Argonaute的身份。总之，我们的结果表明，miRNA降解与Argonaute加载和靶结合竞争，以控制亚细胞miRNA丰度，用于基因沉默监测。探测单个细胞的miRNA活性、运输和代谢有望促进筛选有效的miRNA模拟物和抗miRNA药物。Pitchiaya等人描述了以单分子分辨率询问活细胞内基因调控microRNA的工具。他们发现RNA沉默机制和RNA靶点通过调节microRNA的丰度和亚细胞位置来介导基因沉默监视。这些发现和工具有望促进microRNA活性的单细胞筛选。
Regulation of miRNA localization and stability is critical for their extensive cytoplasmic RNA silencing activity and emerging nuclear functions. Here, we have developed single-molecule fluorescence-based tools to assess the sub-cellular trafficking, integrity and activity of miRNAs. We find that seed-matched RNA targets protect miRNAs against degradation and enhance their nuclear retention. While target-stabilized, functional, cytoplasmic miRNAs reside in high molecular weight complexes, nuclear miRNAs as well as cytoplasmic miRNAs targeted by complementary anti-miRNAs are sequestered stably within significantly lower molecular weight complexes and rendered repression-incompetent. miRNA stability and activity depend on Argonaute protein abundance, whereas miRNA strand selection, unwinding and nuclear retention depend on Argonaute identity. Taken together, our results show that miRNA degradation competes with Argonaute loading and target binding to control sub-cellular miRNA abundance for gene silencing surveillance. Probing single cells for miRNA activity, trafficking and metabolism promises to facilitate screening for effective miRNA mimics and anti-miRNA drugs. Pitchiaya et al. describe tools to interrogate gene-regulatory microRNAs inside living cells at single-molecule resolution. They find that the RNA silencing machinery and RNA targets mediate gene silencing surveillance by modulating abundance and sub-cellular location of microRNAs. These findings and tools promise to facilitate single-cell screening of microRNA activity.