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Affinity-enhanced RNA-binding domains as tools to understand RNA recognition
Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10326445/ https://www.ncbi.nlm.nih.gov/pubmed/37426752 http://dx.doi.org/10.1016/j.crmeth.2023.100508 |
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author | Chaves-Arquero, Belén Collins, Katherine M. Abis, Giancarlo Kelly, Geoff Christodoulou, Evangelos Taylor, Ian A. Ramos, Andres |
author_facet | Chaves-Arquero, Belén Collins, Katherine M. Abis, Giancarlo Kelly, Geoff Christodoulou, Evangelos Taylor, Ian A. Ramos, Andres |
author_sort | Chaves-Arquero, Belén |
collection | PubMed |
description | Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain’s sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains. |
format | Online Article Text |
id | pubmed-10326445 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-103264452023-07-08 Affinity-enhanced RNA-binding domains as tools to understand RNA recognition Chaves-Arquero, Belén Collins, Katherine M. Abis, Giancarlo Kelly, Geoff Christodoulou, Evangelos Taylor, Ian A. Ramos, Andres Cell Rep Methods Report Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain’s sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains. Elsevier 2023-06-26 /pmc/articles/PMC10326445/ /pubmed/37426752 http://dx.doi.org/10.1016/j.crmeth.2023.100508 Text en © 2023 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Report Chaves-Arquero, Belén Collins, Katherine M. Abis, Giancarlo Kelly, Geoff Christodoulou, Evangelos Taylor, Ian A. Ramos, Andres Affinity-enhanced RNA-binding domains as tools to understand RNA recognition |
title | Affinity-enhanced RNA-binding domains as tools to understand RNA recognition |
title_full | Affinity-enhanced RNA-binding domains as tools to understand RNA recognition |
title_fullStr | Affinity-enhanced RNA-binding domains as tools to understand RNA recognition |
title_full_unstemmed | Affinity-enhanced RNA-binding domains as tools to understand RNA recognition |
title_short | Affinity-enhanced RNA-binding domains as tools to understand RNA recognition |
title_sort | affinity-enhanced rna-binding domains as tools to understand rna recognition |
topic | Report |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10326445/ https://www.ncbi.nlm.nih.gov/pubmed/37426752 http://dx.doi.org/10.1016/j.crmeth.2023.100508 |
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