Single Molecule Analysis Group, Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA; Michigan Center for Translational Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-1055, USA; Department of Pathology, University of Michigan, Ann Arbor, MI 48109, USA; Comprehensive Cancer Center, University of Michigan, Ann Arbor, MI 48109, USA.
Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-1055, USA; Consulting for Statistics, Computing and Analytics Research, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Mol Cell. 2019 May 2;74(3):521-533.e6. doi: 10.1016/j.molcel.2019.03.001. Epub 2019 Apr 2.
Cellular RNAs often colocalize with cytoplasmic, membrane-less ribonucleoprotein (RNP) granules enriched for RNA-processing enzymes, termed processing bodies (PBs). Here we track the dynamic localization of individual miRNAs, mRNAs, and long non-coding RNAs (lncRNAs) to PBs using intracellular single-molecule fluorescence microscopy. We find that unused miRNAs stably bind to PBs, whereas functional miRNAs, repressed mRNAs, and lncRNAs both transiently and stably localize within either the core or periphery of PBs, albeit to different extents. Consequently, translation potential and 3' versus 5' placement of miRNA target sites significantly affect the PB localization dynamics of mRNAs. Using computational modeling and supporting experimental approaches, we show that partitioning in the PB phase attenuates mRNA silencing, suggesting that physiological mRNA turnover occurs predominantly outside of PBs. Instead, our data support a PB role in sequestering unused miRNAs for surveillance and provide a framework for investigating the dynamic assembly of RNP granules by phase separation at single-molecule resolution.
细胞 RNA 通常与富含 RNA 加工酶的细胞质、无膜核糖核蛋白 (RNP) 颗粒共定位，这些颗粒被称为处理体 (PBs)。在这里，我们使用细胞内单分子荧光显微镜追踪单个 miRNA、mRNA 和长非编码 RNA (lncRNA) 到 PBs 的动态定位。我们发现未使用的 miRNA 稳定结合到 PBs 上，而功能性 miRNA、被抑制的 mRNA 和 lncRNA 都在 PBs 的核心或外围短暂和稳定地定位，尽管程度不同。因此，miRNA 靶位点的翻译潜力和 3' 端与 5' 端的位置显著影响 mRNA 在 PB 中的定位动力学。通过计算建模和支持性实验方法，我们表明 PB 相的分区会减弱 mRNA 的沉默，这表明生理 mRNA 周转主要发生在 PBs 之外。相反，我们的数据支持 PB 在监视未使用的 miRNA 方面的作用，并提供了一个框架来研究通过相分离在单分子分辨率下动态组装 RNP 颗粒。
