Development of a Sensitive Microplate Assay for Characterizing RNA Methyltransferase Activity: Implications for Epitranscriptomics and Drug Development

Authors

Isaiah K. Mensah
Allison B. Norvil
Ming He
Emma Lendy
Nicole Hjortland
Hern Tan
Richard T. Pomerantz, Thomas Jefferson UniversityFollow
Andrew Mesecar
Humaira Gowher

Document Type

Article

Publication Date

9-14-2023

Comments

This article is the author's final published version in the Journal of Biological Chemistry, Volume 299, Issue 10, October 2023, Article number 105257.

The published version is available at https://doi.org/10.1016/j.jbc.2023.105257. Copyright © 2023 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology

Abstract

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.

Recommended Citation

Mensah, Isaiah K.; Norvil, Allison B.; He, Ming; Lendy, Emma; Hjortland, Nicole; Tan, Hern; Pomerantz, Richard T.; Mesecar, Andrew; and Gowher, Humaira, "Development of a Sensitive Microplate Assay for Characterizing RNA Methyltransferase Activity: Implications for Epitranscriptomics and Drug Development" (2023). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 245.
https://jdc.jefferson.edu/bmpfp/245

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.

PubMed ID

37716702

Language

English