No abstract

Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions, and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress response mechanism involving selective translation of codon-biased mRNA for critical proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-coupled mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA or other RNA species. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS using dynamic multiple reaction monitoring. This approach enables quantification of modified ribonucleosides in several micrograms of tRNA, or other RNA, in a 15-minute LC-MS run. By comparison, traditional methods for detecting modified ribonucleosides are labor and time intensive, require larger RNA quantities, are modification-specific, or require radioactive labeling.

Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo5U in tRNAThr(UGU), which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria.

Acetaminophen (APAP) toxicity is the most common drug-induced cause of acute liver failure in the United States. The only available treatment, N-acetylcysteine (NAC), has a limited time window of efficacy, indicating a need for additional therapeutic options. Zebrafish have emerged as a powerful tool for drug discovery. Here, we developed a clinically relevant zebrafish model of APAP toxicity. APAP depleted glutathione stores, elevated aminotransferase levels, increased apoptosis, and caused dose-dependent hepatocyte necrosis. These outcomes were limited by NAC and conserved in zebrafish embryos. In a targeted embryonic chemical screen, prostaglandin E2 (PGE2) was identified as a potential therapeutic agent; in the adult, PGE2 similarly decreased APAP-associated toxicity. Significantly, when combined with NAC, PGE2 extended the time window for a successful intervention, synergistically reducing apoptosis, improving liver enzymes, and preventing death. Use of a wnt reporter zebrafish line and chemical genetic epistasis showed that the effects of PGE2 are mediated through the wnt signaling pathway. Zebrafish can be used as a clinically relevant toxicological model amenable to the identification of additional therapeutics and biomarkers of APAP injury; our data suggest combinatorial PGE2 and NAC treatment would be beneficial for patients with APAP-induced liver damage.acetaminophen liver toxicity | chemical screen A cetaminophen (N-acetyl-p-aminophenol; APAP) is a commonly used analgesic and antipyretic. Although safe at therapeutic doses, accidental or suicidal drug overdose can cause dosedependent liver damage. APAP is the most common cause for liver transplantation for toxin-induced fulminant hepatic failure and results in more than 300 deaths annually in the United States (1). The only antidote in clinical use is N-acetylcysteine (NAC), which reduces mortality by ∼20-28% (2); however, the time interval of effective intervention after ingestion is typically <12 h, and delayed or prolonged treatment can negatively impact clinical outcome and survival (3). Therapeutic options for hepatic failure are limited to best supportive care and liver transplantation.APAP toxicity results from a hepatotoxic metabolite, N-acetylp-benzoquinone imine (NAPQI), produced by the cytochrome P450 enzymes CYP1A2, 2E1, and 3A4 (4). At therapeutic doses, NAPQI is efficiently inactivated in the liver by glutathione (GSH) conjugation (5). At toxic doses, excess production of NAPQI depletes hepatic GSH. Unconjugated NAPQI causes dysfunction of critical liver proteins, oxidative stress, and mitochondrial damage (6-8). NAC is a true antidote and limits damage by repleting GSH. Clinical efficacy of NAC treatment was shown for patients presenting after APAP ingestion; however, no conclusive clinical trials were conducted to elucidate its efficacy and optimal treatment window. Therapeutic benefits have been shown in mammalian models for other antioxidants (9, 10) that function to restore GSH levels. Compounds that support liver recovery from AP...

Cells respond to stress by controlling gene expression at several levels, with little known about the role of translation. Here, we demonstrate a coordinated translational stress response system involving stress-specific reprogramming of tRNA wobble modifications that leads to selective translation of codon-biased mRNAs representing different classes of critical response proteins. In budding yeast exposed to four oxidants and five alkylating agents, tRNA modification patterns accurately distinguished among chemically similar stressors, with 14 modified ribonucleosides forming the basis for a data-driven model that predicts toxicant chemistry with >80% sensitivity and specificity. tRNA modification subpatterns also distinguish SN1 from SN2 alkylating agents, with SN2-induced increases in m3C in tRNA mechanistically linked to selective translation of threonine-rich membrane proteins from genes enriched with ACC and ACT degenerate codons for threonine. These results establish tRNA modifications as predictive biomarkers of exposure and illustrate a novel regulatory mechanism for translational control of cell stress response.

Post-transcriptional modifications of transfer RNAs (tRNAs) have long been recognized to play crucial roles in regulating the rate and fidelity of translation. However, the extent to which they determine global protein production remains poorly understood. Here we use quantitative proteomics to show a direct link between wobble uridine 5-methoxycarbonylmethyl (mcm5) and 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modifications catalyzed by tRNA methyltransferase 9 (Trm9) in tRNAArg(UCU) and tRNAGlu(UUC) and selective translation of proteins from genes enriched with their cognate codons. Controlling for bias in protein expression and alternations in mRNA expression, we find that loss of Trm9 selectively impairs expression of proteins from genes enriched with AGA and GAA codons under both normal and stress conditions. Moreover, we show that AGA and GAA codons occur with high frequency in clusters along the transcripts, which may play a role in modulating translation. Consistent with these results, proteins subject to enhanced ribosome pausing in yeast lacking mcm5U and mcm5s2U are more likely to be down-regulated and contain a larger number of AGA/GAA clusters. Together, these results suggest that Trm9-catalyzed tRNA modifications play a significant role in regulating protein expression within the cell.

Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. The chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: 1) RNA isolation, 2) RNA purification, 3) RNA hydrolysis to individual ribonucleosides, 4) chromatographic resolution of ribonucleosides, 5) identification of the full set of modified ribonucleosides, 6) mass spectrometric quantification of ribonucleosides, 6) interrogation of ribonucleoside datasets, and 7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.

A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.