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Title: TDP-43 α-helical structure tunes liquid–liquid phase separation and function

Abstract

Liquid–liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix–helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.

Authors:
; ; ; ; ; ; ORCiD logo; ; ; ORCiD logo
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1603181
Grant/Contract Number:  
[DESC0013979]
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
[Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 117 Journal Issue: 11]; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English

Citation Formats

Conicella, Alexander E., Dignon, Gregory L., Zerze, Gül H., Schmidt, Hermann Broder, D’Ordine, Alexandra M., Kim, Young C., Rohatgi, Rajat, Ayala, Yuna M., Mittal, Jeetain, and Fawzi, Nicolas L. TDP-43 α-helical structure tunes liquid–liquid phase separation and function. United States: N. p., 2020. Web. doi:10.1073/pnas.1912055117.
Conicella, Alexander E., Dignon, Gregory L., Zerze, Gül H., Schmidt, Hermann Broder, D’Ordine, Alexandra M., Kim, Young C., Rohatgi, Rajat, Ayala, Yuna M., Mittal, Jeetain, & Fawzi, Nicolas L. TDP-43 α-helical structure tunes liquid–liquid phase separation and function. United States. doi:10.1073/pnas.1912055117.
Conicella, Alexander E., Dignon, Gregory L., Zerze, Gül H., Schmidt, Hermann Broder, D’Ordine, Alexandra M., Kim, Young C., Rohatgi, Rajat, Ayala, Yuna M., Mittal, Jeetain, and Fawzi, Nicolas L. Wed . "TDP-43 α-helical structure tunes liquid–liquid phase separation and function". United States. doi:10.1073/pnas.1912055117.
@article{osti_1603181,
title = {TDP-43 α-helical structure tunes liquid–liquid phase separation and function},
author = {Conicella, Alexander E. and Dignon, Gregory L. and Zerze, Gül H. and Schmidt, Hermann Broder and D’Ordine, Alexandra M. and Kim, Young C. and Rohatgi, Rajat and Ayala, Yuna M. and Mittal, Jeetain and Fawzi, Nicolas L.},
abstractNote = {Liquid–liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix–helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.},
doi = {10.1073/pnas.1912055117},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = [11],
volume = [117],
place = {United States},
year = {2020},
month = {3}
}

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DOI: 10.1073/pnas.1912055117

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