2. Academic Validation
  3. A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing

A single N-terminal phosphomimic disrupts TDP-43 polymerization, phase separation, and RNA splicing

  • EMBO J. 2018 Mar 1;37(5):e97452. doi: 10.15252/embj.201797452.
Ailin Wang 1,  Alexander E Conicella 2,  Hermann Broder Schmidt 3,  Erik W Martin 4,  Shannon N Rhoads 5,  Ashley N Reeb 6,  Amanda Nourse 4,  Daniel Ramirez Montero 1,  Veronica H Ryan 7,  Rajat Rohatgi 3 8,  Frank Shewmaker 5,  Mandar T Naik 1,  Tanja Mittag 4,  Yuna M Ayala 6,  Nicolas L Fawzi 9 2
Affiliations

Affiliations

  • 1 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA.
  • 2 Graduate Program in Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA.
  • 3 Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.
  • 4 Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
  • 5 Department of Pharmacology and Molecular Therapeutics, Uniformed Services University, Bethesda, MD, USA.
  • 6 Edward Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO, USA.
  • 7 Neuroscience Graduate Program, Brown University, Providence, RI, USA.
  • 8 Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA.
  • 9 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI, USA nicolas_fawzi@brown.edu.
PMID: 29438978 DOI: 10.15252/embj.201797452
Abstract

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.

Keywords

RNA splicing; RNP granule; amyotrophic lateral sclerosis; protein–protein interaction; solution NMR spectroscopy.

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