Specific RNA interactions promote TDP-43 multivalent phase separation and maintain liquid properties

TDP-43 is an RNA binding protein that forms ribonucleoprotein condensates via liquid-liquid phase separation (LLPS) and regulates gene expression through specific RNA interactions. Loss of TDP-43 protein homeostasis and dysfunction are tied to neurodegenerative disorders, mainly amyotrophic lateral sclerosis and frontotemporal dementia. Alterations of TDP-43 LLPS properties may be linked to protein aggregation. However, the mechanisms regulating TDP-43 LLPS are ill-defined, particularly how TDP-43 association with specific RNA targets regulates TDP-43 condensation remains unclear. We show that RNA binding strongly promotes TDP-43 LLPS through sequence-specific interactions. RNA-driven condensation increases with the number of adjacent TDP-43 binding sites and is also mediated by multivalent interactions involving the amino and carboxy-terminal TDP-43 domains. The physiological relevance of RNA-driven TDP-43 condensation is supported by similar observations in mammalian cellular lysate. Importantly, we find that TDP-43-RNA association maintains liquid-like properties of the condensates, which are disrupted in the presence of ALS-linked TDP-43 mutations. Altogether, RNA binding plays a central role in modulating TDP-43 condensation while maintaining protein solubility, and defects in this RNA-mediated activity may underpin TDP-43 associated pathogenesis.

TDP-43是一种RNA结合蛋白（RNA binding protein），可通过液-液相分离（liquid-liquid phase separation, LLPS）形成核糖核蛋白凝聚体，并通过特异性RNA相互作用调控基因表达。TDP-43蛋白稳态（protein homeostasis）失衡与功能异常与神经退行性疾病紧密相关，主要包括肌萎缩侧索硬化（amyotrophic lateral sclerosis, ALS）与额颞叶痴呆。TDP-43液-液相分离特性的改变可能与蛋白质聚集（protein aggregation）相关。然而，调控TDP-43液-液相分离的分子机制仍不明确，尤其是TDP-43与特定RNA靶点的结合如何调控其凝聚过程，目前仍不清楚。本研究表明，RNA结合可通过序列特异性相互作用（sequence-specific interactions）显著促进TDP-43的液-液相分离。RNA驱动的凝聚过程随相邻TDP-43结合位点数量的增加而增强，同时也可通过涉及TDP-43氨基末端（amino-terminal）与羧基末端（carboxy-terminal）结构域的多价相互作用（multivalent interactions）介导。在哺乳动物细胞裂解液（mammalian cellular lysate）中观察到的类似现象，佐证了RNA驱动的TDP-43凝聚过程的生理相关性。尤为重要的是，本研究发现TDP-43与RNA的结合可维持凝聚体的液态样特性，而该特性会在携带肌萎缩侧索硬化相关TDP-43突变的样本中被破坏。综上，RNA结合在调控TDP-43凝聚并维持蛋白质可溶性的过程中发挥核心作用，而这一RNA介导的功能缺陷可能是TDP-43相关疾病致病机制（pathogenesis）的核心基础。