细胞内单分子显微镜揭示了 miRNA 组装的两种动力学上不同的途径。
Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly.
机构信息
Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
出版信息
EMBO Rep. 2012 Aug;13(8):709-15. doi: 10.1038/embor.2012.85. Epub 2012 Jun 12.
Abstract
MicroRNAs (miRNAs) associate with components of the RNA-induced silencing complex (RISC) to assemble on mRNA targets and regulate protein expression in higher eukaryotes. Here we describe a method for the intracellular single-molecule, high-resolution localization and counting (iSHiRLoC) of miRNAs. Microinjected, singly fluorophore-labelled, functional miRNAs were tracked within diffusing particles, a majority of which contained single such miRNA molecules. Mobility and mRNA-dependent assembly changes suggest the existence of two kinetically distinct pathways for miRNA assembly, revealing the dynamic nature of this important gene regulatory pathway. iSHiRLOC achieves an unprecedented resolution in the visualization of functional miRNAs, paving the way to understanding RNA silencing through single-molecule systems biology.
摘要
微小 RNA（miRNAs）与 RNA 诱导沉默复合物（RISC）的组成部分结合，组装在 mRNA 靶标上，并在高等真核生物中调节蛋白质表达。在这里，我们描述了一种用于细胞内单分子、高分辨率定位和计数（iSHiRLoC）的 miRNA 的方法。微注射、单荧光标记、功能 miRNA 在扩散颗粒内被追踪，其中大多数颗粒只包含一个这样的 miRNA 分子。迁移率和 mRNA 依赖性组装变化表明 miRNA 组装存在两种动力学上不同的途径，揭示了这条重要基因调控途径的动态性质。iSHiRLOC 在功能 miRNA 的可视化方面达到了前所未有的分辨率，为通过单分子系统生物学理解 RNA 沉默铺平了道路。
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