Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development

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 rad...

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Bibliographic Details
Published in:The Journal of biological chemistry 2023-10, Vol.299 (10), p.105257, Article 105257
Main Authors: Mensah, Isaiah K., Norvil, Allison B., He, Ming, Lendy, Emma, Hjortland, Nicole, Tan, Hern, Pomerantz, Richard T., Mesecar, Andrew, Gowher, Humaira
Format: Article
Language:English
Subjects:
Biological Assay - methods
Biotinylation
biotin–avidin
Drug Development
Epigenesis, Genetic
Gene Expression Profiling
methylation activity
Methyltransferases - metabolism
NS5
Protein Structure, Tertiary
RdRP
RNA
RNA methylation
RNA-Dependent RNA Polymerase - metabolism
Zika virus
Zika Virus - enzymology
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Summary: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.
ISSN:0021-9258
1083-351X
1083-351X
DOI:10.1016/j.jbc.2023.105257