Abstract
The simultaneous detection of all the post-transcriptional modifications (PTMs) that decorate cellular RNA can provide comprehensive information on the effects of changing environmental conditions on the entire epitranscriptome. To capture this type of information, we performed the analysis of ribonucleotide mixtures produced by hydrolysis of total RNA extracts from S. cerevisiae that was grown under hyperosmotic and heat shock conditions. Their global PTM profiles clearly indicated that the cellular responses to these types of stresses involved profound changes in the production of specific PTMs. The observed changes involved not only up-/down-regulation of typical PTMs, but also the outright induction of new ones that were absent under normal conditions, or the elimination of others that were normally present. Pointing toward the broad involvement of different classes of RNAs, many of the newly observed PTMs differed from those engaged in the known tRNA-based mechanism of translational recoding, which is induced by oxidative stress. Some of the expression effects were stress-specific, whereas others were not, thus suggesting that RNA PTMs may perform multifaceted activities in stress response, which are subjected to distinctive regulatory pathways. To explore their signaling networks, we implemented a strategy based on the systematic deletion of genes that connect established response genes with PTM biogenetic enzymes in a putative interactomic map. The results clearly identified PTMs that were under direct HOG control, a well-known protein kinase pathway involved in stress response in eukaryotes. Activation of this signaling pathway has been shown to result in the stabilization of numerous mRNAs and the induction of selected lncRNAs involved in chromatin remodeling. The fact that PTMs are capable of altering the activity of the parent RNAs suggest their possible participation in feedback mechanisms aimed at modulating the regulatory functions of such RNAs. This tantalizing hypothesis will be the object of future studies.
Highlights
From the ‡The RNA Institute, University at Albany (SUNY), Albany, New York 12222; ʈLaboratory of Molecular Genetics, Wadsworth Center, Albany, New York 12208
We subsequently focused on the hyperosmolarity glycerol (HOG) pathway because of the wealth of available information on its role in the hyperosmotic stress response [9, 10], including the well-characterized induction and stabilization of mRNAs and long noncoding RNAs (lncRNAs) [12,13,14,15]
We have recently developed a strategy for obtaining complete post-transcriptional modifications (PTMs) profiles, which involves the analysis of total RNA extracts obtained from cell lysates [34]
Summary
From the ‡The RNA Institute, University at Albany (SUNY), Albany, New York 12222; ʈLaboratory of Molecular Genetics, Wadsworth Center, Albany, New York 12208. Owing to its multifaceted activities in protein synthesis and newly discovered functions in gene regulation, RNA is uniquely positioned among cellular components to act as a communication node between metabolic and regulatory mechanisms of stress response [4, 5]. The transcriptional response to osmostress includes the induction and stabilization of hundreds of mRNAs [12,13,14] and the up-regulation of specific families of long noncoding RNAs (lncRNAs) [15], as well as a general down-regulation of gene expression [15,16,17,18] The latter class of RNA has been shown to induce chromatin remodeling and variations of nucleosome occupancy that have lasting effects on cellular memory and epigenetic makeup [21,22,23]. This approach has been shown capable of revealing significant differences, and subtle variations of PTM expression between samples grown in different media, or obtained altogether from different organisms [34]
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