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

TDP-43 is a nuclear RNA-binding protein regulating numerous steps in RNA metabolism, including alternative splicing. It is a major pathological hallmark of several neurodegenerative diseases, where it forms cytoplasmic aggregates in affected brain regions. TDP-43 can undergo phase separation (PS) and this condensation behavior may be linked to aggregate formation. Whether and how PS governs TDP-43s 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 of TDP-43 variants that form more irreversible, undynamic condensates ("solid-like") in vitro and in cells. Affinity proteomics coupled to mass spectrometry identified a set of interactors whose association with TDP-43 is PS-dependent. This includes multiple splicing regulators and the RNA helicase UPF1, which show increased interactions with solid-like TDP-43 variants. Consistently, we identified PS-dependent alternative splicing events that translate into measurable changes in RNA and protein abundance. Our results highlight that TDP-43 PS regulates RNA and protein homeostasis both directly, by altering a subset of TDP-43-dependent alternative splicing events, and indirectly, by changing interactions with other RNA regulatory factors.