Phase separation-deficient TDP43 remains functional in splicing

Abstract

Intrinsically disordered regions (IDRs) are often fast-evolving protein domains of low sequence complexity that can drive phase transitions and are commonly found in many proteins associated with neurodegenerative diseases, including the RNA processing factor TDP43. Yet, how phase separation contributes to the physiological functions of TDP43 in cells remains enigmatic. Here, we combine systematic mutagenesis guided by evolutionary sequence analysis with a live-cell reporter assay of TDP43 phase dynamics to identify regularly-spaced hydrophobic motifs separated by flexible, hydrophilic segments in the IDR as a key determinant of TDP43 phase properties. This heuristic framework allows customization of the material properties of TDP43 condensates to determine effects on splicing function. Remarkably, even a mutant that fails to phase-separate at physiological concentrations can still efficiently mediate the splicing of a quantitative, single-cell splicing reporter and endogenous targets. This suggests that the ability of TDP43 to phase-separate is not essential for its splicing function. TDP43 regulates RNA processing and undergoes liquid-liquid phase separation. Here, the authors combine bioinformatic analysis and quantitative measurements of phase properties in living cells to uncover the rules that link the amino acid sequence of TDP43 with its phase properties and find that unexpectedly, mutants of TDP43 that are deficient in phase separation can still function to mediate splicing.