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 (ALS) 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. SYNOPSIS Liquid-liquid phase separation of the RNA-binding protein TDP-43 is regulated by specific RNA sequences. In addition to modulating phase separation, RNA is crucial for maintaining the liquid state of TDP-43 condensates. GU-repeat RNA specifically promotes TDP-43 liquid-liquid phase separation and this increases with multiple binding sites. Binding of specific RNA sequences to the TDP-43 RNA-binding domains maintains liquidity of TDP-43 condensates. Both the N- and C-terminal TDP-43 domains are required for RNA-driven liquid-liquid phase separation, constituting a multivalent network. The liquid properties of RNA-driven TDP-43 condensates are disrupted in a subset of ALS-linked TDP-43 mutations. Graphical Abstract Liquid-liquid phase separation of the RNA-binding protein TDP-43 is regulated by specific RNA sequences. In addition to modulating phase separation, RNA is crucial for maintaining the liquid state of TDP-43 condensates.