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EMBO J. 2018 Mar 1;37(5). pii: e97452. doi: 10.15252/embj.201797452. Epub 2018 Feb 9.

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

Author information

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.

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

PMID:
29438978
PMCID:
PMC5830921
DOI:
10.15252/embj.201797452
[Indexed for MEDLINE]
Free PMC Article

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