PolyG RNA Induces Phase Separation and Precipitation of TLS/FUS

Translocated in Liposarcoma (TLS), also known as Fused in Sarcoma (FUS), is a multifunctional RNA-binding protein implicated in neurodegenerative diseases due to its tendency to aggregate. While mutations in TLS are linked to familial amyotrophic lateral sclerosis (ALS), approximately 90% of ALS cases are sporadic with no known genetic mutations. In these instances, pathological aggregation of wild-type TLS is believed to play a critical role, although the molecular triggers remain elusive. RNA is known to modulate TLS phase separation, but the features that drive RNA-induced precipitation are poorly understood. Here, we report that synthetic PolyG RNA robustly induces both phase separation and irreversible precipitation of recombinant TLS in vitro. This effect is concentration-dependent and strongly influenced by RNA sequence composition. Specifically, guanine-rich RNAs such as PolyG promote aggregation, whereas uridine-rich RNAs fail to induce precipitation and may even inhibit it. These findings suggest a selective interaction between TLS and G-rich RNA sequences. Notably, the resulting TLS-RNA complexes undergo precipitation in a manner distinct from classical liquid-liquid phase separation, highlighting a unique mechanism of RNA-induced protein misfolding. Through detailed molecular biological and biochemical analyses, we further demonstrate that PolyG-induced condensates transition into solid-like aggregates over time. Our results uncover a previously uncharacterized pathway of RNA-mediated TLS aggregation and suggest that guanine-rich RNAs may contribute to pathological protein misfolding in neurodegenerative disease contexts.