Phase separation behavior of TDP-43 governs its protein interactome and regulation of alternative splicing

TDP-43 is a nuclear RNA-binding protein that regulates RNA metabolism, including alternative splicing. Its aggregation is a major pathological hallmark of several neurodegenerative diseases. TDP-43 undergoes phase separation (PS) and this condensation behavior may be linked to aggregate formation. Whether and how PS governs TDP-43’s RNA regulatory functions remains poorly understood. Here we utilized rationally designed mutations in the TDP-43 low complexity domain to tune TDP-43 PS, yielding a panel of TDP-43 variants with reduced propensity to form condensates (“PS-deficient”), and a panel forming irreversible, undynamic condensates (“solid-like”) in vitro and in cells. Two complementary interactomics approaches identified PS-dependent interactions between TDP-43 and key RNA regulatory factors, including splicing regulators and the RNA helicase UPF1, which show increased interactions with solid-like variants. Our results highlight that TDP-43 PS regulates RNA and protein homeostasis by modulating a subset of TDP-43-dependent alternative splicing events and by reshaping interactions with RNA regulatory factors.