Journal
EMBO JOURNAL
Volume 37, Issue 5, Pages -Publisher
WILEY
DOI: 10.15252/embj.201797452
Keywords
amyotrophic lateral sclerosis; protein-protein interaction; RNA splicing; RNP granule; solution NMR spectroscopy
Categories
Funding
- National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (NIH) [R01GM118530, R35GM119790, R01GM112846]
- Institutional Development Award (IDeA) from NIGMS [P20GM104937]
- ALS Association [17-IIP-342]
- ALS Research grant from the Judith AMP
- Jean Pape Adams Charitable Foundation
- American Lebanese Syrian Associated Charities
- NIGMS [T32GM07601, P30GM103410]
- NIMH [T32MH020068]
- Brown Institute for Brain Science Graduate Awards
- NIH [DP2GM105448, R35GM118082]
- Deutsche Forschungsgemeinschaft [SCHM 3082/2-1]
- Division of Biology and Medicine, Brown University
- NCRR [P30RR031153, P20RR018728, S10RR02763]
- National Science Foundation EPSCoR [0554548]
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TDP-43 is an RNA-binding protein active in splicing that concentrates into membraneless ribonucleoprotein granules and forms aggregates in amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Although best known for its predominantly disordered C- terminal domain which mediates ALS inclusions, TDP-43 has a globular N-terminal domain (NTD). Here, we show that TDP-43 NTD assembles into head-to-tail linear chains and that phosphomimetic substitution at S48 disrupts TDP-43 polymeric assembly, discourages liquid-liquid phase separation (LLPS) in vitro, fluidizes liquid-liquid phase separated nuclear TDP-43 reporter constructs in cells, and disrupts RNA splicing activity. Finally, we present the solution NMR structure of a head-to-tail NTD dimer comprised of two engineered variants that allow saturation of the native polymerization interface while disrupting higher-order polymerization. These data provide structural detail for the established mechanistic role of the well-folded TDP-43 NTD in splicing and link this function to LLPS. In addition, the fusion-tag solubilized, recombinant form of TDP-43 full-length protein developed here will enable future phase separation and in vitro biochemical assays on TDP-43 function and interactions that have been hampered in the past by TDP-43 aggregation.
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