Adivarahan Srivathsan, Zenklusen Daniel
Département de biochimie et médecine moléculaire, Université de Montréal, Montréal, QC, Canada.
Methods Mol Biol. 2021;2209:267-286. doi: 10.1007/978-1-0716-0935-4_17.
mRNAs and lncRNAs assemble with RNA-binding proteins (RBPs) to form ribonucleoprotein complexes (RNPs ). The assembly of RNPs initiates co-transcriptionally, and their composition and organization is thought to change during the different steps of an RNP life cycle. Modulation of RNP structural organization has been implicated in the regulation of different aspects of RNA metabolism, including establishing interactions between the 5' and 3' ends in regulating mRNA translation and turnover. In this chapter, we describe a single-molecule microscopy approach that combines fluorescent RNA in situ hybridization (smFISH) and structured illumination microscopy (SIM ) and allows to measure different aspects of RNP organization in cells, including distances between different regions within individual mRNAs, as well as the overall compaction state of RNAs in different subcellular compartments and environmental conditions. Moreover, we describe a detailed workflow required for image registration and analysis that allows determining distances at sub-diffraction resolution.
信使核糖核酸（mRNAs）和长链非编码核糖核酸（lncRNAs）与核糖核酸结合蛋白（RBPs）组装形成核糖核蛋白复合体（RNPs）。核糖核蛋白复合体的组装在转录过程中起始，并且其组成和结构被认为在核糖核蛋白复合体生命周期的不同阶段会发生变化。核糖核蛋白复合体结构组织的调控与RNA代谢的不同方面有关，包括在调节信使核糖核酸翻译和周转过程中建立5'端和3'端之间的相互作用。在本章中，我们描述了一种单分子显微镜方法，该方法结合了荧光RNA原位杂交（smFISH）和结构照明显微镜（SIM），能够测量细胞中核糖核蛋白复合体组织的不同方面，包括单个信使核糖核酸内不同区域之间的距离，以及不同亚细胞区室和环境条件下RNA的整体压缩状态。此外，我们描述了图像配准和分析所需的详细工作流程，该流程能够在亚衍射分辨率下确定距离。
