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.
Highlights
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
Even though amino-acid substitutions in the C-terminal domain (CTD) have been frequent during vertebrate evolution, point mutations that lead to single amino-acid changes and cause familial amyotrophic lateral sclerosis (ALS) in humans are most frequently found in the CTD21,22, highlighting the importance of decoding the sequence rules underlying TDP43 phase behavior in physiology and disease
Phase transitions of proteins and nucleic acids into biomolecular condensates in cells are emerging as an important cellular mechanism to dynamically organize biochemical reactions in space and time[51,52,53]
Summary
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. The CTD harbors most known human mutations in TDP43 that cause ALS16 Some of these mutations have been shown to alter the material properties of TDP43 condensates[15,17] and to interfere with TDP43-mediated splicing[18], implying that TDP43 phase separation may be required for the function of TDP43 in RNA processing. Even mutations that abrogated TDP43 phase transitions at physiological concentrations failed to prevent the TDP43-dependent splicing of endogenous targets or a quantitative single-cell splicing reporter These findings suggest that condensate formation is not required for the function of TDP43 in exon skipping
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