RNA structural probing of guanine and uracil nucleotides in yeast

• Type of Material: Article
• Authors: Kevin Xiao, Emory University; Homa Ghalei, Emory University; Sohail Khoshnevis, Emory University
• Language: English
• Date: 2023-07-07
• Publisher: PUBLIC LIBRARY SCIENCE
• Publication Version: Final Published Version
• Copyright Statement: © 2023 Xiao et al
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0288070
• Title of Journal or Parent Work: PLOS ONE
• Volume: 18
• Issue: 7
• Start Page: e0288070
• End Page: e0288070
• Grant/Funding Information: This work was supported by Emory University Research Council (URC) grant to S.K. and NIH grant 1R35GM138123 to H.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
• Keywords: Multidisciplinary Sciences; Science & Technology - Other Topics; Science & Technology; PROBES
• Research Categories: Biology, Microbiology; Chemistry, General; Chemistry, Biochemistry

Abstract

RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-pToluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.