Abstract
During their biosynthesis, transfer RNAs (tRNAs) are decorated with a large number of posttranscriptional chemical modifications. Methods to directly detect the introduction of posttranscriptional modifications during tRNA maturation are rare and do not provide information on the temporality of modification events. Here, we report a methodology, using NMR as a tool to monitor tRNA maturation in a nondisruptive and continuous fashion in cellular extracts. This method requires the production of substrate tRNA transcripts devoid of modifications and active cell extracts containing the necessary cellular enzymatic activities to modify RNA. The present protocol describes these different aspects of our method and reports the time-resolved NMR monitoring of the yeast tRNAPhe maturation as an example. The NMR-based methodology presented here could be adapted to investigate diverse features in tRNA maturation.
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Acknowledgments
The authors are grateful to Henri Grosjean for protocols and stimulating discussions about RNA modifications, Sylvie Auxilien (I2BC) for the c13-ABYS-86 yeast strain, Bruno Sargueil for guidance regarding the Eaton press implementation, and Christel Le Bon for ensuring the best performance of the NMR infrastructure at the IBPC. The authors acknowledge access to the biomolecular NMR platform of the IBPC that is supported by the CNRS, the Labex DYNAMO (ANR-11-LABX-0011), the Equipex CACSICE (ANR-11-EQPX-0008), and the Conseil Régional d’Île-de-France (SESAME grant). This work was supported by grant ANR-14-CE09-0012 from the ANR.
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Gato, A., Catala, M., Tisné, C., Barraud, P. (2021). A Method to Monitor the Introduction of Posttranscriptional Modifications in tRNAs with NMR Spectroscopy. In: McMahon, M. (eds) RNA Modifications. Methods in Molecular Biology, vol 2298. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1374-0_19
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DOI: https://doi.org/10.1007/978-1-0716-1374-0_19
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