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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2021
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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. |
format | Online Article Text |
id | pubmed-8373067 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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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