Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
Institute for Chemical Reaction Design and Discovery, Hokkaido University, Sapporo, Japan.
Methods Mol Biol. 2022;2509:361-393. doi: 10.1007/978-1-0716-2380-0_22.
Phase separation is one of the fundamental processes to compartmentalize biomolecules in living cells. RNA-protein complexes (RNPs) often scaffold biomolecular condensates formed through phase separation. We here present a statistical thermodynamics approach to investigate intracellular phase separation. We first present the statistical thermodynamic theory of the liquid-liquid phase separation (LLPS) of two molecules (such as proteins and solvent molecules) and of a polymer solution (such as RNPs and solvent molecules). Condensates produced by LLPS show coarsening and/or coalescence to minimize their total surface area. In addition to the LLPS, there are other types of self-assembly, such as microphase separation, micellization, emulsification, and vesiculation, with which the growth of the assembly stops with optimal size and shape. We also describe a scaling theory of micelles of block copolymers, where their structures are analogous to the core-shell structure of paraspeckle nuclear bodies scaffolded by RNPs of NEAT1_2 long noncoding RNAs (lncRNAs) and RNA-binding proteins (RBPs). These theories treat the self-assembly of polymers in the thermodynamic equilibrium, where their concentrations and compositions do not change with time. In contrast, RNPs are produced according to the transcription of RNAs and are degraded with time. We therefore take into account the dynamical aspect of the production of RNPs in an extension of the theory of the self-assembly of soft matter. Finally, we discuss the structure of paraspeckles as an example to demonstrate that an approach combining experiment and theory is powerful to investigate the mechanism of intracellular phase separation.
相分离是将生物分子在活细胞中分隔成区室的基本过程之一。RNA-蛋白质复合物（RNP）通常作为支架，形成通过相分离形成的生物分子凝聚物。我们在这里提出了一种统计热力学方法来研究细胞内的相分离。我们首先介绍了两种分子（如蛋白质和溶剂分子）和聚合物溶液（如 RNP 和溶剂分子）的液-液相分离（LLPS）的统计热力学理论。LLPS 产生的凝聚物会粗化和/或聚结，以最小化它们的总表面积。除了 LLPS 之外，还有其他类型的自组装，如微相分离、胶束化、乳化和囊泡化，这些自组装的生长会停止在最佳的大小和形状。我们还描述了嵌段共聚物胶束的标度理论，其中它们的结构类似于由 NEAT1_2 长非编码 RNA（lncRNA）和 RNA 结合蛋白（RBP）的 RNP 支架的核体 paraspeckle 的核壳结构。这些理论将聚合物的自组装处理为热力学平衡中的状态，其中它们的浓度和组成不会随时间变化。相比之下，RNP 根据 RNA 的转录产生，并随时间降解。因此，我们在软物质自组装理论的扩展中考虑了 RNP 产生的动态方面。最后，我们讨论了 paraspeckles 的结构，作为一个例子，演示了结合实验和理论的方法对于研究细胞内相分离机制的强大性。
