2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions
Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological...
| Main Authors: | , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2024-02-01
|
| Series: | Frontiers in Molecular Biosciences |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2024.1325041/full |
| _version_ | 1827351385726779392 |
|---|---|
| author | Hesna Kara Hesna Kara Alexander Axer Frederick W. Muskett Frederick W. Muskett Carlos J. Bueno-Alejo Carlos J. Bueno-Alejo Vasileios Paschalis Vasileios Paschalis Andrea Taladriz-Sender Sumera Tubasum Sumera Tubasum Marina Santana Vega Zhengyun Zhao Alasdair W. Clark Andrew J. Hudson Andrew J. Hudson Ian C. Eperon Ian C. Eperon Glenn A. Burley Cyril Dominguez Cyril Dominguez |
| author_facet | Hesna Kara Hesna Kara Alexander Axer Frederick W. Muskett Frederick W. Muskett Carlos J. Bueno-Alejo Carlos J. Bueno-Alejo Vasileios Paschalis Vasileios Paschalis Andrea Taladriz-Sender Sumera Tubasum Sumera Tubasum Marina Santana Vega Zhengyun Zhao Alasdair W. Clark Andrew J. Hudson Andrew J. Hudson Ian C. Eperon Ian C. Eperon Glenn A. Burley Cyril Dominguez Cyril Dominguez |
| author_sort | Hesna Kara |
| collection | DOAJ |
| description | Protein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2′ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts. |
| first_indexed | 2024-03-08T02:02:28Z |
| format | Article |
| id | doaj.art-2051e50dad5a4945b5af04fd03395abe |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-03-08T02:02:28Z |
| publishDate | 2024-02-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-2051e50dad5a4945b5af04fd03395abe2024-02-14T04:53:56ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2024-02-011110.3389/fmolb.2024.132504113250412′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditionsHesna Kara0Hesna Kara1Alexander Axer2Frederick W. Muskett3Frederick W. Muskett4Carlos J. Bueno-Alejo5Carlos J. Bueno-Alejo6Vasileios Paschalis7Vasileios Paschalis8Andrea Taladriz-Sender9Sumera Tubasum10Sumera Tubasum11Marina Santana Vega12Zhengyun Zhao13Alasdair W. Clark14Andrew J. Hudson15Andrew J. Hudson16Ian C. Eperon17Ian C. Eperon18Glenn A. Burley19Cyril Dominguez20Cyril Dominguez21Department of Molecular and Cellular Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomWestCHEM and Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United KingdomDepartment of Molecular and Cellular Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomSchool of Chemistry, University of Leicester, Leicester, United KingdomDepartment of Molecular and Cellular Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomWestCHEM and Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United KingdomDepartment of Molecular and Cellular Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomBiomedical Engineering Research Division, School of Engineering, University of Glasgow, Glasgow, United KingdomWestCHEM and Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United KingdomBiomedical Engineering Research Division, School of Engineering, University of Glasgow, Glasgow, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomSchool of Chemistry, University of Leicester, Leicester, United KingdomDepartment of Molecular and Cellular Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomWestCHEM and Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow, United KingdomDepartment of Molecular and Cellular Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomLeicester Institute of Structural and Chemical Biology, Henry Wellcome Building, University of Leicester, Leicester, United KingdomProtein-RNA interactions are central to numerous cellular processes. In this work, we present an easy and straightforward NMR-based approach to determine the RNA binding site of RNA binding proteins and to evaluate the binding of pairs of proteins to a single-stranded RNA (ssRNA) under physiological conditions, in this case in nuclear extracts. By incorporation of a 19F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the 19F NMR signal changes when the 19F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two 19F atoms at two different locations informs on their concurrent binding or competition. We demonstrate that such studies can be done in a nuclear extract that mimics the physiological environment in which these protein-ssRNA interactions occur. Finally, we demonstrate that a trifluoromethoxy group (-OCF3) incorporated in the 2′ribose position of ssRNA sequences increases the sensitivity of the NMR signal, leading to decreased measurement times, and reduces the issue of RNA degradation in cellular extracts.https://www.frontiersin.org/articles/10.3389/fmolb.2024.1325041/fullRNA-protein interaction19F NMR spectroscopyRNA binding proteinsRNA labellingconcurrent/competitive binding |
| spellingShingle | Hesna Kara Hesna Kara Alexander Axer Frederick W. Muskett Frederick W. Muskett Carlos J. Bueno-Alejo Carlos J. Bueno-Alejo Vasileios Paschalis Vasileios Paschalis Andrea Taladriz-Sender Sumera Tubasum Sumera Tubasum Marina Santana Vega Zhengyun Zhao Alasdair W. Clark Andrew J. Hudson Andrew J. Hudson Ian C. Eperon Ian C. Eperon Glenn A. Burley Cyril Dominguez Cyril Dominguez 2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions Frontiers in Molecular Biosciences RNA-protein interaction 19F NMR spectroscopy RNA binding proteins RNA labelling concurrent/competitive binding |
| title | 2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions |
| title_full | 2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions |
| title_fullStr | 2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions |
| title_full_unstemmed | 2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions |
| title_short | 2′-19F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions |
| title_sort | 2 19f labelling of ribose in rnas a tool to analyse rna protein interactions by nmr in physiological conditions |
| topic | RNA-protein interaction 19F NMR spectroscopy RNA binding proteins RNA labelling concurrent/competitive binding |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2024.1325041/full |
| work_keys_str_mv | AT hesnakara 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT hesnakara 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT alexanderaxer 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT frederickwmuskett 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT frederickwmuskett 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT carlosjbuenoalejo 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT carlosjbuenoalejo 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT vasileiospaschalis 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT vasileiospaschalis 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT andreataladrizsender 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT sumeratubasum 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT sumeratubasum 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT marinasantanavega 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT zhengyunzhao 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT alasdairwclark 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT andrewjhudson 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT andrewjhudson 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT ianceperon 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT ianceperon 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT glennaburley 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT cyrildominguez 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions AT cyrildominguez 219flabellingofriboseinrnasatooltoanalysernaproteininteractionsbynmrinphysiologicalconditions |