Disease-associated mutations affect TIA1 phase separation and aggregation in a proline-dependent manner.

T-cell restriction intracellular antigen 1 (TIA1) is an RNA-binding protein that is a major component of stress granules (SGs). The low complexity domain (LCD) of TIA1 plays a central role in facilitating SGs assembly through liquid-liquid phase separation (LLPS). Disruption of the LLPS process has been associated with several diseases. It has recently been shown that the proline-rich domain affects the LLPS process of some proteins (such as UBQLN2 and Tau). Thus, proline may regulate LLPS. The LCD of TIA1 contains 11 proline residues, and several proline-related mutations have been shown to cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we demonstrated that TIA1 can undergo phase separation in cells. Additionally, disease-associated proline-to-leucine (P-L) mutations, which altered droplet morphology, facilitated the liquid-to-solid phase transition of TIA1 into solid-like amyloid fibrils. The changes in the physical properties of the P-L mutation altered the behavior of TIA1 in vivo and led to abnormal SGs kinetics, resulting in the formation of the pathological inclusions of ALS. Prolines are the key residues for regulating the LLPS of TIA1.