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The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization

The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 bind...

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Autores principales: Yan, Kevin Kok-Phen, Obi, Ikenna, Sabouri, Nasim
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373067/
https://www.ncbi.nlm.nih.gov/pubmed/34302476
http://dx.doi.org/10.1093/nar/gkab620
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author Yan, Kevin Kok-Phen
Obi, Ikenna
Sabouri, Nasim
author_facet Yan, Kevin Kok-Phen
Obi, Ikenna
Sabouri, Nasim
author_sort Yan, Kevin Kok-Phen
collection PubMed
description The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization.
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spelling pubmed-83730672021-08-19 The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization Yan, Kevin Kok-Phen Obi, Ikenna Sabouri, Nasim Nucleic Acids Res Nucleic Acid Enzymes The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization. Oxford University Press 2021-07-24 /pmc/articles/PMC8373067/ /pubmed/34302476 http://dx.doi.org/10.1093/nar/gkab620 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) ), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Nucleic Acid Enzymes
Yan, Kevin Kok-Phen
Obi, Ikenna
Sabouri, Nasim
The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization
title The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization
title_full The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization
title_fullStr The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization
title_full_unstemmed The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization
title_short The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization
title_sort rgg domain in the c-terminus of the dead box helicases dbp2 and ded1 is necessary for g-quadruplex destabilization
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373067/
https://www.ncbi.nlm.nih.gov/pubmed/34302476
http://dx.doi.org/10.1093/nar/gkab620
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