Molecular Coping Mechanisms: Reprogramming tRNAs To Regulate Codon-Biased Translation of Stress Response Proteins.

As part of the classic central dogma of molecular biology, transfer RNAs (tRNAs) are integral to protein translation as the adaptor molecules that link the genetic code in messenger RNA (mRNA) to the amino acids in the growing peptide chain. tRNA function is complicated by the existence of 61 codons to specify 20 amino acids, with most amino acids coded by two or more synonymous codons. Further, there are often fewer tRNAs with unique anticodons than there are synonymous codons for an amino acid, with a single anticodon able to decode several codons by "wobbling" of the base pairs arising between the third base of the codon and the first position on the anticodon. The complications introduced by synonymous codons and wobble base pairing began to resolve in the 1960s with the discovery of dozens of chemical modifications of the ribonucleotides in tRNA, which, by analogy to the epigenome, are now collectively referred to as the epitranscriptome for not changing the genetic code inherent to all RNA sequences. tRNA modifications were found to stabilize codon-anticodon interactions, prevent misinitiation of translation, and promote translational fidelity, among other functions, with modification deficiencies causing pathological phenotypes. This led to hypotheses that modification-dependent tRNA decoding efficiencies might play regulatory roles in cells. However, it was only with the advent of systems biology and convergent "omic" technologies that the higher level function of synonymous codons and tRNA modifications began to emerge.Here, we describe our laboratories' discovery of tRNA reprogramming and codon-biased translation as a mechanism linking tRNA modifications and synonymous codon usage to regulation of gene expression at the level of translation. Taking a historical approach, we recount how we discovered that the 8-10 modifications in each tRNA molecule undergo unique reprogramming in response to cellular stresses to promote translation of mRNA transcripts with unique codon usage patterns. These modification tunable transcripts (MoTTs) are enriched with specific codons that are differentially decoded by modified tRNAs and that fall into functional families of genes encoding proteins necessary to survive the specific stress. By developing and applying systems-level technologies, we showed that cells lacking specific tRNA modifications are sensitized to certain cellular stresses by mistranslation of proteins, disruption of mitochondrial function, and failure to translate critical stress response proteins. In essence, tRNA reprogramming serves as a cellular coping strategy, enabling rapid translation of proteins required for stress-specific cell response programs. Notably, this phenomenon has now been characterized in all organisms from viruses to humans and in response to all types of environmental changes. We also elaborate on recent findings that cancer cells hijack this mechanism to promote their own growth, metastasis, and chemotherapeutic resistance. We close by discussing how understanding of codon-biased translation in various systems can be exploited to develop new therapeutics and biomanufacturing processes.

Dysfunctional tRNA reprogramming and codon-biased translation in cancer.

Many cancers hijack translation to increase the synthesis of tumor-driving proteins, the messenger mRNAs of which have specific codon usage patterns. Termed 'codon-biased translation' and originally identified in stress response regulation, this mechanism is supported by diverse studies demonstrating how the 50 RNA modifications of the epitranscriptome, specific tRNAs, and codon-biased mRNAs are used by oncogenic programs to promote proliferation and chemoresistance. The epitranscriptome writers METTL1-WDR4, Elongator complex protein (ELP)1-6, CTU1-2, and ALKBH8-TRM112 illustrate the principal mechanism of codon-biased translation, with gene amplifications, increased RNA modifications, and enhanced tRNA stability promoting cancer proliferation. Furthermore, systems-level analyses of 34 tRNA writers and 493 tRNA genes highlight the theme of tRNA epitranscriptome dysregulation in many cancers and identify candidate tRNA writers, tRNA modifications, and tRNA molecules as drivers of pathological codon-biased translation.

Arsenite toxicity is regulated by queuine availability and oxidation-induced reprogramming of the human tRNA epitranscriptome.

Cells respond to environmental stress by regulating gene expression at the level of both transcription and translation. The ∼50 modified ribonucleotides of the human epitranscriptome contribute to the latter, with mounting evidence that dynamic regulation of transfer RNA (tRNA) wobble modifications leads to selective translation of stress response proteins from codon-biased genes. Here we show that the response of human hepatocellular carcinoma cells to arsenite exposure is regulated by the availability of queuine, a micronutrient and essential precursor to the wobble modification queuosine (Q) on tRNAs reading GUN codons. Among oxidizing and alkylating agents at equitoxic concentrations, arsenite exposure caused an oxidant-specific increase in Q that correlated with up-regulation of proteins from codon-biased genes involved in energy metabolism. Limiting queuine increased arsenite-induced cell death, altered translation, increased reactive oxygen species levels, and caused mitochondrial dysfunction. In addition to demonstrating an epitranscriptomic facet of arsenite toxicity and response, our results highlight the links between environmental exposures, stress tolerance, RNA modifications, and micronutrients.

Disease-associated inosine misincorporation into RNA hinders translation.

Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.

Selenoproteins and the senescence-associated epitranscriptome.

Selenium is a naturally found trace element, which provides multiple benefits including antioxidant, anticancer, and antiaging, as well as boosting immunity. One unique feature of selenium is its incorporation as selenocysteine, a rare 21st amino acid, into selenoproteins. Twenty-five human selenoproteins have been discovered, and a majority of these serve as crucial antioxidant enzymes for redox homeostasis. Unlike other amino acids, incorporation of selenocysteine requires a distinctive UGA stop codon recoding mechanism. Although many studies correlating selenium, selenoproteins, aging, and senescence have been performed, it has not yet been explored if the upstream events regulating selenoprotein synthesis play a role in senescence-associated pathologies. The epitranscriptomic writer alkylation repair homolog 8 (ALKBH8) is critical for selenoprotein production, and its deficiency can significantly decrease levels of selenoproteins that are essential for reactive oxygen species (ROS) detoxification, and increase oxidative stress, one of the major drivers of cellular senescence. Here, we review the potential role of epitranscriptomic marks that govern selenocysteine utilization in regulating the senescence program.

Detecting the epitranscriptome.

RNA modifications and their corresponding epitranscriptomic writer and eraser enzymes regulate gene expression. Altered RNA modification levels, dysregulated writers, and sequence changes that disrupt epitranscriptomic marks have been linked to mitochondrial and neurological diseases, cancer, and multifactorial disorders. The detection of epitranscriptomics marks is challenging, but different next generation sequencing (NGS)-based and mass spectrometry-based approaches have been used to identify and quantitate the levels of individual and groups of RNA modifications. NGS and mass spectrometry-based approaches have been coupled with chemical, antibody or enzymatic methodologies to identify modifications in most RNA species, mapped sequence contexts and demonstrated the dynamics of specific RNA modifications, as well as the collective epitranscriptome. While epitranscriptomic analysis is currently limited to basic research applications, specific approaches for the detection of individual RNA modifications and the epitranscriptome have potential biomarker applications in detecting human conditions and diseases. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Processing > tRNA Processing RNA in Disease and Development > RNA in Disease.

Quantitative mapping of the cellular small RNA landscape with AQRNA-seq.

Current next-generation RNA-sequencing (RNA-seq) methods do not provide accurate quantification of small RNAs within a sample, due to sequence-dependent biases in capture, ligation and amplification during library preparation. We present a method, absolute quantification RNA-sequencing (AQRNA-seq), that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member microRNA reference library, oligonucleotide standards of varying length, and RNA blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial transfer RNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced, tRNA-driven, codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells.

Queuine Is a Nutritional Regulator of Entamoeba histolytica Response to Oxidative Stress and a Virulence Attenuator.

Queuosine is a naturally occurring modified ribonucleoside found in the first position of the anticodon of the transfer RNAs for Asp, Asn, His, and Tyr. Eukaryotes lack pathways to synthesize queuine, the nucleobase precursor to queuosine, and must obtain it from diet or gut microbiota. Here, we describe the effects of queuine on the physiology of the eukaryotic parasite Entamoeba histolytica, the causative agent of amebic dysentery. Queuine is efficiently incorporated into E. histolytica tRNAs by a tRNA-guanine transglycosylase (EhTGT) and this incorporation stimulates the methylation of C38 in [Formula: see text] Queuine protects the parasite against oxidative stress (OS) and antagonizes the negative effect that oxidation has on translation by inducing the expression of genes involved in the OS response, such as heat shock protein 70 (Hsp70), antioxidant enzymes, and enzymes involved in DNA repair. On the other hand, queuine impairs E. histolytica virulence by downregulating the expression of genes previously associated with virulence, including cysteine proteases, cytoskeletal proteins, and small GTPases. Silencing of EhTGT prevents incorporation of queuine into tRNAs and strongly impairs methylation of C38 in [Formula: see text], parasite growth, resistance to OS, and cytopathic activity. Overall, our data reveal that queuine plays a dual role in promoting OS resistance and reducing parasite virulence.IMPORTANCEEntamoeba histolytica is a unicellular parasite that causes amebiasis. The parasite resides in the colon and feeds on the colonic microbiota. The gut flora is implicated in the onset of symptomatic amebiasis due to alterations in the composition of bacteria. These bacteria modulate the physiology of the parasite and affect the virulence of the parasite through unknown mechanisms. Queuine, a modified nucleobase of queuosine, is exclusively produced by the gut bacteria and leads to tRNA modification at the anticodon loops of specific tRNAs. We found that queuine induces mild oxidative stress resistance in the parasite and attenuates its virulence. Our study highlights the importance of bacterially derived products in shaping the physiology of the parasite. The fact that queuine inhibits the virulence of E. histolytica may lead to new strategies for preventing and/or treating amebiasis by providing to the host queuine directly or via probiotics.

The epitranscriptomic writer ALKBH8 drives tolerance and protects mouse lungs from the environmental pollutant naphthalene.

The epitranscriptomic writer Alkylation Repair Homolog 8 (ALKBH8) is a transfer RNA (tRNA) methyltransferase that modifies the wobble uridine of selenocysteine tRNA to promote the specialized translation of selenoproteins. Using Alkbh8 deficient (Alkbh8def) mice, we have investigated the importance of epitranscriptomic systems in the response to naphthalene, an abundant polycyclic aromatic hydrocarbon and environmental toxicant. We performed basal lung analysis and naphthalene exposure studies using wild type (WT), Alkbh8def and Cyp2abfgs-null mice, the latter of which lack the cytochrome P450 enzymes required for naphthalene bioactivation. Under basal conditions, lungs from Alkbh8def mice have increased markers of oxidative stress and decreased thioredoxin reductase protein levels, and have reprogrammed gene expression to differentially regulate stress response transcripts. Alkbh8def mice are more sensitive to naphthalene induced death than WT, showing higher susceptibility to lung damage at the cellular and molecular levels. Further, WT mice develop a tolerance to naphthalene after 3 days, defined as resistance to a high challenging dose after repeated exposures, which is absent in Alkbh8def mice. We conclude that the epitranscriptomic writer ALKBH8 plays a protective role against naphthalene-induced lung dysfunction and promotes naphthalene tolerance. Our work provides an early example of how epitranscriptomic systems can regulate the response to environmental stress in vivo.

Loss of Elongator- and KEOPS-Dependent tRNA Modifications Leads to Severe Growth Phenotypes and Protein Aggregation in Yeast.

Modifications found in the Anticodon Stem Loop (ASL) of tRNAs play important roles in regulating translational speed and accuracy. Threonylcarbamoyl adenosine (t6A37) and 5-methoxycarbonyl methyl-2-thiouridine (mcm5s2U34) are critical ASL modifications that have been linked to several human diseases. The model yeast Saccharomyces cerevisiae is viable despite the absence of both modifications, growth is however greatly impaired. The major observed consequence is a subsequent increase in protein aggregates and aberrant morphology. Proteomic analysis of the t6A-deficient strain (sua5 mutant) revealed a global mistranslation leading to protein aggregation without regard to physicochemical properties or t6A-dependent or biased codon usage in parent genes. However, loss of sua5 led to increased expression of soluble proteins for mitochondrial function, protein quality processing/trafficking, oxidative stress response, and energy homeostasis. These results point to a global function for t6A in protein homeostasis very similar to mcm5/s2U modifications.

Loss of epitranscriptomic control of selenocysteine utilization engages senescence and mitochondrial reprogramming☆.

Critically important to the maintenance of the glutathione (GSH) redox cycle are the activities of many selenocysteine-containing GSH metabolizing enzymes whose translation is controlled by the epitranscriptomic writer alkylation repair homolog 8 (ALKBH8). ALKBH8 is a tRNA methyltransferase that methylates the wobble uridine of specific tRNAs to regulate the synthesis of selenoproteins. Here we demonstrate that a deficiency in the writer ALKBH8 (Alkbh8def), alters selenoprotein levels and engages senescence, regulates stress response genes and promotes mitochondrial reprogramming. Alkbh8def mouse embryonic fibroblasts (MEFs) increase many hallmarks of senescence, including senescence associated ß-galactosidase, heterocromatic foci, the cyclin dependent kinase inhibitor p16Ink4a, markers of mitochondrial dynamics as well as the senescence associated secretory phenotype (SASP). Alkbh8def cells also acquire a stress resistance phenotype that is accompanied by an increase in a number redox-modifying transcripts. In addition, Alkbh8def MEFs undergo a metabolic shift that is highlighted by a striking increase in the level of uncoupling protein 2 (UCP2) which enhances oxygen consumption and promotes a reliance on glycolytic metabolism. Finally, we have shown that the Alkbh8 deficiency can be exploited and corresponding MEFs are killed by glycolytic inhibition. Our work demonstrates that defects in an epitransciptomic writer promote senescence and mitochondrial reprogramming and unveils a novel adaptive mechanism for coping with defects in selenocysteine utilization.

Epitranscriptomic systems regulate the translation of reactive oxygen species detoxifying and disease linked selenoproteins.

Here we highlight the role of epitranscriptomic systems in post-transcriptional regulation, with a specific focus on RNA modifying writers required for the incorporation of the 21st amino acid selenocysteine during translation, and the pathologies linked to epitranscriptomic and selenoprotein defects. Epitranscriptomic marks in the form of enzyme-catalyzed modifications to RNA have been shown to be important signals regulating translation, with defects linked to altered development, intellectual impairment, and cancer. Modifications to rRNA, mRNA and tRNA can affect their structure and function, while the levels of these dynamic tRNA-specific epitranscriptomic marks are stress-regulated to control translation. The tRNA for selenocysteine contains five distinct epitranscriptomic marks and the ALKBH8 writer for the wobble uridine (U) has been shown to be vital for the translation of the glutathione peroxidase (GPX) and thioredoxin reductase (TRXR) family of selenoproteins. The reactive oxygen species (ROS) detoxifying selenocysteine containing proteins are a prime examples of how specialized translation can be regulated by specific tRNA modifications working in conjunction with distinct codon usage patterns, RNA binding proteins and specific 3' untranslated region (UTR) signals. We highlight the important role of selenoproteins in detoxifying ROS and provide details on how epitranscriptomic marks and selenoproteins can play key roles in and maintaining mitochondrial function and preventing disease.

Phosphorylation of human TRM9L integrates multiple stress-signaling pathways for tumor growth suppression.

The human transfer RNA methyltransferase 9-like gene (TRM9L, also known as KIAA1456) encodes a negative regulator of tumor growth that is frequently silenced in many forms of cancer. While TRM9L can inhibit tumor cell growth in vivo, the molecular mechanisms underlying the tumor inhibition activity of TRM9L are unknown. We show that oxidative stress induces the rapid and dose-dependent phosphorylation of TRM9L within an intrinsically disordered domain that is necessary for tumor growth suppression. Multiple serine residues are hyperphosphorylated in response to oxidative stress. Using a chemical genetic approach, we identified a key serine residue in TRM9L that undergoes hyperphosphorylation downstream of the oxidative stress-activated MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase)-RSK (ribosomal protein S6 kinase) signaling cascade. Moreover, we found that phosphorylated TRM9L interacts with the 14-3-3 family of proteins, providing a link between oxidative stress and downstream cellular events involved in cell cycle control and proliferation. Mutation of the serine residues required for TRM9L hyperphosphorylation and 14-3-3 binding abolished the tumor inhibition activity of TRM9L. Our results uncover TRM9L as a key downstream effector of the ERK signaling pathway and elucidate a phospho-signaling regulatory mechanism underlying the tumor inhibition activity of TRM9L.

Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease.

The rise of advanced technologies for characterizing human populations at the molecular level, from sequence to function, is shifting disease prevention paradigms toward personalized strategies. Because minimization of adverse outcomes is a key driver for treatment decisions for diseased populations, developing personalized therapy strategies represent an important dimension of both precision medicine and personalized prevention. In this commentary, we highlight recently developed enabling technologies in the field of DNA damage, DNA repair, and mutagenesis. We propose that omics approaches and functional assays can be integrated into population studies that fuse basic, translational and clinical research with commercial expertise in order to accelerate personalized prevention and treatment of cancer and other diseases linked to aberrant responses to DNA damage. This collaborative approach is generally applicable to efforts to develop data-driven, individualized prevention and treatment strategies for other diseases. We also recommend strategies for maximizing the use of biological samples for epidemiological studies, and for applying emerging technologies to clinical applications.

Mitochondrial ROS control of cancer.

Mitochondria serves a primary role in energy maintenance but also function to govern levels of mitochondria-derived reactive oxygen species (mROS). ROS have long been established to play a critical role in tumorigenesis and are now considered to be integral to the regulation of diverse signaling networks that drive proliferation, tumor cell survival and malignant progression. mROS can damage DNA, activate oncogenes, block the function of tumor suppressors and drive migratory signaling. The mitochondrion's oxidant scavenging systems including SOD2, Grx2, GPrx, Trx and TrxR are key of the cellular redox tone. These mitochondrial antioxidant systems serve to tightly control the levels of the primary ROS signaling species, H2O2. The coordinated control of mROS levels is also coupled to the activity of the primary H2O2 consuming enzymes of the mitochondria which are reliant on the epitranscriptomic control of selenocysteine incorporation. This review highlights the interplay between these many oncogenic signaling networks, mROS and the H2O2 emitting and consuming capacity of the mitochondria.

tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence.

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.

Comparative analysis of redox and inflammatory properties of pristine nanomaterials and commonly used semiconductor manufacturing nano-abrasives.

Continued expansion of the nanotechnology industry has necessitated the self-assessment of manufacturing processes, specifically in regards to understanding the health related aspects following exposure to nanomaterials. There exists a growing concern over potential occupational exposure in the semiconductor industry where Al2O3, CeO2 and SiO2 nanoparticles are commonly featured as part of the chemical mechanical planarization (CMP) process. Chronic exposure to toxicants can result not only in acute cytotoxicity but also initiation of a chronic inflammatory state associated with diverse pathologies. In the current investigation, pristine nanoparticles and CMP slurry formulations of Al2O3, SiO2 and CeO2 were employed to assess their ability to induce cytotoxicity, inflammatory responses and reactive oxygen species in a mouse alveolar macrophage cell model. The pristine nanoparticles and slurries were not intrinsically cytotoxic and did not generate free radicals but were found to act as scavengers in the presence of an oxidant stimulant. Al2O3 and SiO2 nanoparticles increased levels of pro-inflammatory cytokines while pristine SiO2 nanoparticles induced generation of F2-Isoprostanes. In co-treatment studies, the pristine nanomaterials modulated the response to the inflammatory stimulant lipopolysaccharide. The studies have established that pristine nanoparticles and slurries do not impact the cells in a similar way indicating that they should not be used as slurry substitutes in toxicity evaluations. Further, we have defined how an alveolar cell line, which would likely be the first challenged upon nanomaterial aerosolization, responds to diverse mixtures of nanomaterials. Moreover, our findings reinforce the importance of using multiple analytic methods to define the redox state of the cell following exposure to commonly used industrial nanomaterials and toxicants.

Alkbh8 Regulates Selenocysteine-Protein Expression to Protect against Reactive Oxygen Species Damage.

Environmental and metabolic sources of reactive oxygen species (ROS) can damage DNA, proteins and lipids to promote disease. Regulation of gene expression can prevent this damage and can include increased transcription, translation and post translational modification. Cellular responses to ROS play important roles in disease prevention, with deficiencies linked to cancer, neurodegeneration and ageing. Here we detail basal and damage-induced translational regulation of a group of oxidative-stress response enzymes by the tRNA methyltransferase Alkbh8. Using a new gene targeted knockout mouse cell system, we show that Alkbh8-/- embryonic fibroblasts (MEFs) display elevated ROS levels, increased DNA and lipid damage and hallmarks of cellular stress. We demonstrate that Alkbh8 is induced in response to ROS and is required for the efficient expression of selenocysteine-containing ROS detoxification enzymes belonging to the glutathione peroxidase (Gpx1, Gpx3, Gpx6 and likely Gpx4) and thioredoxin reductase (TrxR1) families. We also show that, in response to oxidative stress, the tRNA modification 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm5Um) increases in normal MEFs to drive the expression of ROS detoxification enzymes, with this damage-induced reprogramming of tRNA and stop-codon recoding corrupted in Alkbh8-/- MEFS. These studies define Alkbh8 and tRNA modifications as central regulators of cellular oxidative stress responses in mammalian systems. In addition they highlight a new animal model for use in environmental and cancer studies and link translational regulation to the prevention of DNA and lipid damage.

Codon-biased translation can be regulated by wobble-base tRNA modification systems during cellular stress responses.

tRNA (tRNA) is a key molecule used for protein synthesis, with multiple points of stress-induced regulation that can include transcription, transcript processing, localization and ribonucleoside base modification. Enzyme-catalyzed modification of tRNA occurs at a number of base and sugar positions and has the potential to influence specific anticodon-codon interactions and regulate translation. Notably, altered tRNA modification has been linked to mitochondrial diseases and cancer progression. In this review, specific to Eukaryotic systems, we discuss how recent systems-level analyses using a bioanalytical platform have revealed that there is extensive reprogramming of tRNA modifications in response to cellular stress and during cell cycle progression. Combined with genome-wide codon bias analytics and gene expression studies, a model emerges in which stress-induced reprogramming of tRNA drives the translational regulation of critical response proteins whose transcripts display a distinct codon bias. Termed Modification Tunable Transcripts (MoTTs), (1) we define them as (1) transcripts that use specific degenerate codons and codon biases to encode critical stress response proteins, and (2) transcripts whose translation is influenced by changes in wobble base tRNA modification. In this review we note that the MoTTs translational model is also applicable to the process of stop-codon recoding for selenocysteine incorporation, as stop-codon recoding involves a selective codon bias and modified tRNA to decode selenocysteine during the translation of a key subset of oxidative stress response proteins. Further, we discuss how in addition to RNA modification analytics, the comprehensive characterization of translational regulation of specific transcripts requires a variety of tools, including high coverage codon-reporters, ribosome profiling and linked genomic and proteomic approaches. Together these tools will yield important new insights into the role of translational elongation in cell stress response.

Gcn5 Modulates the Cellular Response to Oxidative Stress and Histone Deacetylase Inhibition.

To identify chemical genetic interactions underlying the mechanism of action of histone deacetylase inhibitors (HDACi) a yeast deletion library was screened for hypersensitive deletion mutants that confer increased sensitivity to the HDACi, CG-1521. The screen demonstrated that loss of GCN5 or deletion of components of the Gcn5 histone acetyltransferase (HAT) complex, SAGA, sensitizes yeast to CG-1521-induced cell death. Expression profiling after CG-1521 treatment reveals increased expression of genes involved in metabolism and oxidative stress response, and oxidative stress response mutants are hypersensitive to CG-1521 treatment. Accumulation of reactive oxygen species and increased cell death are enhanced in the gcn5Δ deletion mutant, and are abrogated by anti-oxidants, indicating a central role of oxidative stress in CG-1521-induced cell death. In human cell lines, siRNA mediated knockdown of GCN5 or PCAF, or chemical inhibition of GCN5 enzymatic activity, increases the sensitivity to CG-1521 and SAHA. These data suggest that the combination of HDAC and GCN5/PCAF inhibitors can be used for cancer treatment.