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

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 inclu...

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Autores principales: Wang, Ailin, Conicella, Alexander E, Schmidt, Hermann Broder, Martin, Erik W, Rhoads, Shannon N, Reeb, Ashley N, Nourse, Amanda, Ramirez Montero, Daniel, Ryan, Veronica H, Rohatgi, Rajat, Shewmaker, Frank, Naik, Mandar T, Mittag, Tanja, Ayala, Yuna M, Fawzi, Nicolas L
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830921/
https://www.ncbi.nlm.nih.gov/pubmed/29438978
http://dx.doi.org/10.15252/embj.201797452
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author Wang, Ailin
Conicella, Alexander E
Schmidt, Hermann Broder
Martin, Erik W
Rhoads, Shannon N
Reeb, Ashley N
Nourse, Amanda
Ramirez Montero, Daniel
Ryan, Veronica H
Rohatgi, Rajat
Shewmaker, Frank
Naik, Mandar T
Mittag, Tanja
Ayala, Yuna M
Fawzi, Nicolas L
author_facet Wang, Ailin
Conicella, Alexander E
Schmidt, Hermann Broder
Martin, Erik W
Rhoads, Shannon N
Reeb, Ashley N
Nourse, Amanda
Ramirez Montero, Daniel
Ryan, Veronica H
Rohatgi, Rajat
Shewmaker, Frank
Naik, Mandar T
Mittag, Tanja
Ayala, Yuna M
Fawzi, Nicolas L
author_sort Wang, Ailin
collection PubMed
description 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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spelling pubmed-58309212018-03-14 A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing Wang, Ailin Conicella, Alexander E Schmidt, Hermann Broder Martin, Erik W Rhoads, Shannon N Reeb, Ashley N Nourse, Amanda Ramirez Montero, Daniel Ryan, Veronica H Rohatgi, Rajat Shewmaker, Frank Naik, Mandar T Mittag, Tanja Ayala, Yuna M Fawzi, Nicolas L EMBO J Articles 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. John Wiley and Sons Inc. 2018-02-09 2018-03-01 /pmc/articles/PMC5830921/ /pubmed/29438978 http://dx.doi.org/10.15252/embj.201797452 Text en © 2018 The Authors. Published under the terms of the CC BY 4.0 license This is an open access article under the terms of the Creative Commons Attribution 4.0 (http://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Articles
Wang, Ailin
Conicella, Alexander E
Schmidt, Hermann Broder
Martin, Erik W
Rhoads, Shannon N
Reeb, Ashley N
Nourse, Amanda
Ramirez Montero, Daniel
Ryan, Veronica H
Rohatgi, Rajat
Shewmaker, Frank
Naik, Mandar T
Mittag, Tanja
Ayala, Yuna M
Fawzi, Nicolas L
A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing
title A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing
title_full A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing
title_fullStr A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing
title_full_unstemmed A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing
title_short A single N‐terminal phosphomimic disrupts TDP‐43 polymerization, phase separation, and RNA splicing
title_sort single n‐terminal phosphomimic disrupts tdp‐43 polymerization, phase separation, and rna splicing
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830921/
https://www.ncbi.nlm.nih.gov/pubmed/29438978
http://dx.doi.org/10.15252/embj.201797452
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