2′-19F labelling of ribose in RNAs: a tool to analyse RNA/Protein interactions by NMR in physiological conditions

Kara, H. et al. (2024) 2′-19F labelling of ribose in RNAs: a tool to analyse RNA/Protein interactions by NMR in physiological conditions. Frontiers in Molecular Biosciences, 11, 1325041. (doi: 10.3389/fmolb.2024.1325041) (PMID:38419689) (PMCID:PMC10899400)

Abstract

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.

• Item Type: Articles
• Additional Information: This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) sLoLa grant (BB/T000627/1) to AC, AH, GB, IE, and CD
• Keywords: RNA-protein interaction, 19F NMR spectroscopy, RNA binding proteins, RNA labelling.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Santana Vega, Dr Marina and Clark, Professor Alasdair

Creator Roles

Santana Vega, M.: Methodology, Writing – review and editing
Clark, A.: Funding acquisition, Methodology, Project administration, Writing – review and editing

• Authors: Kara, H., Axer, A., Muskett, F. W., Bueno-Alejo, C. J., Paschalis, V., Taladriz-Sender, A., Tubasum, S., Santana Vega, M., Zhao, Z., Clark, A. W., Hudson, A. J., Eperon, I. C., Burley, G. A., and Dominguez, C.
• College/School: College of Science and Engineering > School of Engineering > Biomedical Engineering
• Journal Name: Frontiers in Molecular Biosciences
• Publisher: Frontiers Media
• ISSN: 2296-889X
• ISSN (Online): 2296-889X
• Copyright Holders: Copyright © 2024 Kara, Axer, Muskett, Bueno-Alejo, Paschalis, Taladriz-Sender, Tubasum, Vega, Zhao, Clark, Hudson, Eperon, Burley and Dominguez
• First Published: First published in Frontiers in Molecular Biosciences 11:1325041
• Publisher Policy: Reproduced under a Creative Commons license