Roy Raju, Rajyaguru Purusharth I
Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India.
Bio Protoc. 2022 Aug 20;12(16). doi: 10.21769/BioProtoc.4487.
RNA granules are conserved, non-membranous, biphasic structures predominantly composed of RNA and RNA-binding proteins. RNA granules often assemble as a result of cellular responses to a variety of stresses, including infection. Two types of RNA granules are best characterized: stress granules (SGs) and processing bodies (P-bodies). The mechanism of RNA granule assembly and disassembly is still understudied because of its complex composition and dynamic behavior. The assembly of RNA granules is driven by a process known as phase separation of granule components. Edc3 is a conserved decapping activator and an essential P-body component in Phase separation of P-body proteins has been poorly explored. This protocol will enable the visualization of the phase transition behavior of Edc3, since it is tagged to mCherry. It further describes using small molecules and other proteins to study P-body dynamics. In addition to the assembly of Edc3, this assay also lays the foundation to study disassembly of phase-separated assemblies , which was not explored earlier. We have devised the assay to describe the use of one such protein that acts as a disassembly factor. Overall, this protocol is simple to perform and can potentially be combined with analyzing these assemblies using other approaches. Graphical abstract.
RNA颗粒是保守的、无膜的双相结构，主要由RNA和RNA结合蛋白组成。RNA颗粒通常是细胞对包括感染在内的各种应激反应的结果。两种类型的RNA颗粒特征最为明显：应激颗粒（SGs）和加工小体（P小体）。由于RNA颗粒的组成复杂且行为动态，其组装和拆卸机制仍未得到充分研究。RNA颗粒的组装由一种称为颗粒成分相分离的过程驱动。Edc3是一种保守的去帽激活剂，也是P小体的重要组成成分。P小体蛋白的相分离研究较少。该方案将能够可视化Edc3的相变行为，因为它被标记为mCherry。它还进一步描述了使用小分子和其他蛋白质来研究P小体动力学。除了Edc3的组装，该分析方法还为研究相分离组装体的拆卸奠定了基础，而这在之前并未被探索过。我们设计了该分析方法来描述一种作为拆卸因子的蛋白质的使用。总体而言，该方案操作简单，并且有可能与使用其他方法分析这些组装体相结合。图形摘要。
