RNA structural probing of guanine and uracil nucleotides in yeast

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 r...

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Bibliographic Details
Published in:PloS one 2023-07, Vol.18 (7), p.e0288070-e0288070
Main Authors: Xiao, Kevin, Ghalei, Homa, Khoshnevis, Sohail
Format: Article
Language:English
Subjects:
Adenine
Analysis
Biocompatibility
Biology and Life Sciences
Carbodiimide
Cells
Cellular structure
Dimethyl sulfate
Effectiveness
Gene expression
Genetic aspects
Guanine
In vivo methods and tests
Investigations
Medicine and Health Sciences
Nucleotides
Pathogens
Phenols
Properties
Pyrimidines
Research and Analysis Methods
Ribonucleic acid
RNA
RNA processing
RNA-protein interactions
Saccharomyces cerevisiae
Structure-function relationships
Sulfates
Uracil
Yeast
Yeast fungi
Yeasts
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Summary: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-p-Toluenesulfonate (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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0288070