Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response

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.

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“…RNA concentrations were determined by using UV 260 nm and adjusted to 40 ng/l. The RNA was then treated with nuclease P1 and phosphodiesterase to obtain the desired NMP mixtures for mass spectrometric analysis, as previously described (49,50). It is important to note that this analytical platform is capable of correctly discriminating NMPs from dNMPs based on their masses and fragmentation properties.…”

“…Immediately before analysis, the mononucleotide mixtures obtained by exonuclease digestion of intact RNA were diluted to 4 ng/l in 150 mM ammonium acetate and 10% isopropanol. All samples were analyzed on a Thermo Scientific LTQ-Orbitrap Velos instrument and a Waters Synapt G2 HDMS ion mobility spectrometry mass spectrometer (IMS-MS), as previously described (49,50). Analyses were accomplished by using direct infusion electrospray ionization (ESI) in negative ion mode.…”

“…Tandem mass spectrometry was carried out in both positive and negative mode to differentiate possible isobars detected during PTM analysis (49,50). This approach involves recognizing unique fragments produced by gas-phase activation, which reveal the position of modifying functional groups.…”

“…We performed a comprehensive investigation of PTMs on total RNA extracted directly from cell lysates, that is without fractionating the RNA into the individual classes of RNAs (see 'Materials and Methods' sec-tion). More specifically, total RNA was isolated by performing TRIzol extraction, DNase 1 digestion and then ethanol precipitation to eliminate unwanted DNA, as well as any pre-existing mononucleotides and soluble components, which may have been present in the initial extract (49,50). The intact RNA was then hydrolyzed into mononucleotide mixtures with specific exonucleases, and analyzed by directinfusion nanoflow ESI (nanoESI, 61,62) mass spectrometry (see 'Materials and Methods' section).…”

“…Indeed, MS analysis has been instrumental to the discovery of all known PTMs listed in the RNA Modification Database (4,5). Based on this platform, we have recently developed a robust approach for an agnostic analysis of PTMs at the whole transcriptome level (49,50). This comprehensive approach can reveal the effects of different environmental factors on the expression of the entire complement of modifications, including variations of distinct subsets contained in the cell, and thus enables the investigation of RNA modifications at a systems level (50).…”