RNAs, across their numerous classes, often work in concert with proteins in RNA-protein complexes (RNPs) to execute critical cellular functions. Ensemble-averaging methods have been instrumental in revealing many important aspects of these RNA-protein interactions, yet are insufficiently sensitive to much of the dynamics at the heart of RNP function. Single-molecule fluorescence microscopy (SMFM) offers complementary, versatile tools to probe RNP conformational and compositional changes in detail. In this review, we first outline the basic principles of SMFM as applied to RNPs, describing key considerations for labeling, imaging, and quantitative analysis. We then sample applications of in vitro and in vivo single-molecule visualization using the case studies of pre-messenger RNA (mRNA) splicing and RNA silencing, respectively. After discussing specific insights single-molecule fluorescence methods have yielded, we briefly review recent developments in the field and highlight areas of anticipated growth.

中文翻译：

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聚集在一起：RNA和蛋白质在单分子荧光显微镜下组装。

RNA在其众多类别中通常与RNA-蛋白质复合物（RNP）中的蛋白质协同工作，以执行关键的细胞功能。集成平均法已在揭示这些RNA蛋白质相互作用的许多重要方面发挥了作用，但对RNP功能核心的许多动力学不够敏感。单分子荧光显微镜（SMFM）提供了互补的多功能工具，可详细探测RNP的构象和组成变化。在这篇综述中，我们首先概述了SMFM应用于RNP的基本原理，描述了标记，成像和定量分析的主要考虑因素。然后，我们分别使用信使前RNA（mRNA）剪接和RNA沉默的案例研究，对体外和体内单分子可视化的应用进行采样。