1. 2'- 19 F labelling of ribose in RNAs: a tool to analyse RNA/protein interactions by NMR in physiological conditions.
- Author
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Kara H, Axer A, Muskett FW, Bueno-Alejo CJ, Paschalis V, Taladriz-Sender A, Tubasum S, Vega MS, Zhao Z, Clark AW, Hudson AJ, Eperon IC, Burley GA, and Dominguez C
- 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
19 F atom on the ribose of different nucleotides along the ssRNA sequence, we show that, upon addition of an RNA binding protein, the intensity of the19 F NMR signal changes when the19 F atom is located near the protein binding site. Furthermore, we show that the addition of pairs of proteins to a ssRNA containing two19 F 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Kara, Axer, Muskett, Bueno-Alejo, Paschalis, Taladriz-Sender, Tubasum, Vega, Zhao, Clark, Hudson, Eperon, Burley and Dominguez.)- Published
- 2024
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