in vivo RNA structural probing of guanine and uracil nucleotides in yeast

Abstract

RNAs have critical catalytic or regulatory functions in the cell and play significant roles in many steps of gene expression regulation. 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 structural probing is an indirect method to probe the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. The chemical modifications can target either the RNA backbone or the Watson-Crick face of nucleotides. The selective 2-hydroxyl acylation analyzed by primer extension (SHAPE) can probe the ribose sugar in all unpaired RNA nucleotides. In contrast, Dimethyl sulfate (DMS) alkylates adenine and cytosine and reports on base pairing context but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in the plant model system Oryza sativa and in the prokaryotic organisms Bacillus subtilis and Echerichia coli, as well as human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyzed the effectiveness of guanine modification by a family of aldehyde derivatives, the glyoxal family, in Saccharomyces cerevisiae and Candida albicans. We also explored the effectiveness of uracil modification by carbodiimide N-cyclohexyl-N-(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate (CMCT) in vivo. We show that among the glyoxal family, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. We also demonstrate uracil modification by CMCT in S. cerevisiae in vivo. 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. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.