A System of RNA Modifications and Biased Codon Use Controls Cellular Stress Response at the Level of Translation

Cells respond to environmental stressors and xenobiotic exposures using regulatory networks to control gene expression, and there is an emerging appreciation for the role of numerous postsynthetic chemical modifications of DNA, RNA, and proteins in controlling transcription and translation of the stress response. In this Perspective, we present a model for a new network that regulates the cellular response to xenobiotic exposures and other stresses in which stress-induced reprogramming of a system of dozens of post-transcriptional modifications on tRNA (tRNA) promotes selective translation of codon-biased mRNAs for critical response proteins. As a product of novel genomic and bioanalytical technologies, this model has strong parallels with the regulatory networks of DNA methylation in epigenetics and the variety of protein secondary modifications comprising signaling pathways and the histone code. When present at the tRNA wobble position, the modified ribonucleosides enhance the translation of mRNAs in which the cognate codons of the tRNAs are highly over-represented and that represent critical stress response proteins. A parallel system may also downregulate the translation of families of proteins. Notably, dysregulation of the tRNA methyltransferase enzymes in humans has also been implicated in cancer etiology, with demonstrated oncogenic and tumor-suppressive effects.National Institutes of Health (U.S.) (NIH ES017010)National Science Foundation (U.S.) (CHE-1308839)Singapore. National Research Foundatio

tRNA modifications regulate translation during cellular stress

The regulation of gene expression in response to stress is an essential cellular protection mechanism. Recent advances in tRNA modification analysis and genome-based codon bias analytics have facilitated studies that lead to a novel model for translational control, with translation elongation dynamically regulated during stress responses. Stress-induced increases in specific anticodon wobble bases are required for the optimal translation of stress response transcripts that are significantly biased in the use of degenerate codons keyed to these modified tRNA bases. These findings led us to introduce the notion of tRNA modification tunable transcripts (MoTTs – transcripts whose translation is regulated by tRNA modifications), which are identifiable using genome-wide codon counting algorithms. In support of this general model of translational control of stress response, studies making use of detailed measures of translation, tRNA methyltransferase mutants, and computational and mass spectrometry approaches reveal that stress reprograms tRNA modifications to translationally regulate MoTTs linked to arginine and leucine codons, which helps cells survive insults by damaging agents. These studies highlight how tRNA methyltransferase activities and MoTTs are key components of the cellular stress response.National Science Foundation (U.S.) (CHE-1308839)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology Center. Infectious Disease Research Program)David H. Koch Cancer Research Fund (Graduate Fellowship)Howard Hughes Medical Institute (International Student Research Fellowship

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), 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.National Science Foundation (U.S.) (CHE-1308839)Singapore. National Research Foundation (Singapore-MIT Alliance for Research and Technology Center. Infectious Disease Research Program

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-methyluridi​ne(mcm[superscript 5]Um) 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[superscript -/-] 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.National Institute of Environmental Health Sciences (ES002109)National Institute of Environmental Health Sciences (ES015037)National Institute of Environmental Health Sciences (ES017010)Westaway Research FundDavid H. Koch Institute for Integrative Cancer Research at MIT (Cancer Research Graduate Fellowship)Howard Hughes Medical Institute (Graduate Fellowship)Singapore-MIT Alliance for Research and Technolog

Human AlkB Homolog ABH8 Is a tRNA Methyltransferase Required for Wobble Uridine Modification and DNA Damage Survival

tRNA nucleosides are extensively modified to ensure their proper function in translation. However, many of the enzymes responsible for tRNA modifications in mammals await identification. Here, we show that human AlkB homolog 8 (ABH8) catalyzes tRNA methylation to generate 5-methylcarboxymethyl uridine (mcm[superscript 5]U) at the wobble position of certain tRNAs, a critical anticodon loop modification linked to DNA damage survival. We find that ABH8 interacts specifically with tRNAs containing mcm5U and that purified ABH8 complexes methylate RNA in vitro. Significantly, ABH8 depletion in human cells reduces endogenous levels of mcm[superscript 5]U in RNA and increases cellular sensitivity to DNA-damaging agents. Moreover, DNA-damaging agents induce ABH8 expression in an ATM-dependent manner. These results expand the role of mammalian AlkB proteins beyond that of direct DNA repair and support a regulatory mechanism in the DNA damage response pathway involving modulation of tRNA modification.United States. National Institutes of Health (grant CA055042)United States. National Institutes of Health (grant ES002109)United States. National Institutes of Health (grant ES01701)National Institutes of Health (U.S.). Intramural Research ProgramWestaway Research FundNational Center for Research Resources (U.S.) (grant S10-RR023783

A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-α

Emerging evidence points to aberrant regulation of translation as a driver of cell transformation in cancer. Given the direct control of translation by tRNA modifications, tRNA modifying enzymes may function as regulators of cancer progression. Here, we show that a tRNA methyltransferase 9‐like (hTRM9L/KIAA1456) mRNA is down‐regulated in breast, bladder, colorectal, cervix and testicular carcinomas. In the aggressive SW620 and HCT116 colon carcinoma cell lines, hTRM9L is silenced and its re‐expression and methyltransferase activity dramatically suppressed tumour growth in vivo. This growth inhibition was linked to decreased proliferation, senescence‐like G0/G1‐arrest and up‐regulation of the RB interacting protein LIN9. Additionally, SW620 cells re‐expressing hTRM9L did not respond to hypoxia via HIF1‐α‐dependent induction of GLUT1. Importantly, hTRM9L‐negative tumours were highly sensitive to aminoglycoside antibiotics and this was associated with altered tRNA modification levels compared to antibiotic resistant hTRM9L‐expressing SW620 cells. Our study links hTRM9L and tRNA modifications to inhibition of tumour growth via LIN9 and HIF1‐α‐dependent mechanisms. It also suggests that aminoglycoside antibiotics may be useful to treat hTRM9L‐deficient tumours.National Institute of Environmental Health Sciences (R01 ES015037)National Institute of Environmental Health Sciences (R01 ES017010)National Institute of Environmental Health Sciences (R21 ES017146)National Institute of Environmental Health Sciences (P30 ES002109)National Cancer Institute (U.S.) (R01 CA109182)National Cancer Institute (U.S.) (U54 CA163131)National Science Foundation (U.S.) (NSF 0922830)NYSTARWestaway Research FundSingapore-MIT Alliance for Research and TechnologySamuel Waxman Cancer Research Foundation Tumour Dormancy ProgramNYSTE

Increased tRNA modification and gene-specific codon usage regulate cell cycle progression during the DNA damage response

S-phase and DNA damage promote increased ribonucleotide reductase (RNR) activity. Translation of RNR1 has been linked to the wobble uridine modifying enzyme tRNA methyltransferase 9 (Trm9). We predicted that changes in tRNA modification would translationally regulate RNR1 after DNA damage to promote cell cycle progression. In support, we demonstrate that the Trm9-dependent tRNA modification 5-methoxycarbonylmethyluridine (mcm⁵U) is increased in hydroxyurea (HU)-induced S-phase cells, relative to G₁ and G₂, and that mcm⁵U is one of 16 tRNA modifications whose levels oscillate during the cell cycle. Codon-reporter data matches the mcm⁵U increase to Trm9 and the efficient translation of AGA codons and RNR1. Further, we show that in trm9Δ cells reduced Rnr1 protein levels cause delayed transition into S-phase after damage. Codon re-engineering of RNR1 increased the number of trm9Δ cells that have transitioned into S-phase 1 h after DNA damage and that have increased Rnr1 protein levels, similar to that of wild-type cells expressing native RNR1. Our data supports a model in which codon usage and tRNA modification are regulatory components of the DNA damage response, with both playing vital roles in cell cycle progression.National Institute of Environmental Health Sciences (R01 ES015037)National Institute of Environmental Health Sciences (R01 ES017010)National Institute of Environmental Health Sciences (P30 ES002109)Massachusetts Institute of Technology (Westaway Fund)Singapore-MIT Alliance for Research and Technolog

The DNA-damage signature in Saccharomyces cerevisiae is associated with single-strand breaks in DNA

BACKGROUND: Upon exposure to agents that damage DNA, Saccharomyces cerevisiae undergo widespread reprogramming of gene expression. Such a vast response may be due not only to damage to DNA but also damage to proteins, RNA, and lipids. Here the transcriptional response of S. cerevisiae specifically induced by DNA damage was discerned by exposing S. cerevisiae to a panel of three "radiomimetic" enediyne antibiotics (calicheamicin γ(1)(I), esperamicin A1 and neocarzinostatin) that bind specifically to DNA and generate varying proportions of single- and double-strand DNA breaks. The genome-wide responses were compared to those induced by the non-selective oxidant γ-radiation. RESULTS: Given well-controlled exposures that resulted in similar and minimal cell death (~20–25%) across all conditions, the extent of gene expression modulation was markedly different depending on treatment with the enediynes or γ-radiation. Exposure to γ-radiation resulted in more extensive transcriptional changes classified both by the number of genes modulated and the magnitude of change. Common biological responses were identified between the enediynes and γ-radiation, with the induction of DNA repair and stress response genes, and the repression of ribosomal biogenesis genes. Despite these common responses, a fraction of the response induced by gamma radiation was repressed by the enediynes and vise versa, suggesting that the enediyne response is not entirely "radiomimetic." Regression analysis identified 55 transcripts with gene expression induction associated both with double- or single-strand break formation. The S. cerevisiae "DNA damage signature" genes as defined by Gasch et al. [1] were enriched among regulated transcripts associated with single-strand breaks, while genes involved in cell cycle regulation were associated with double-strand breaks. CONCLUSION: Dissection of the transcriptional response in yeast that is specifically signaled by DNA strand breaks has identified that single-strand breaks provide the signal for activation of transcripts encoding proteins involved in the DNA damage signature in S. cerevisiae, and double-strand breaks signal changes in cell cycle regulation genes

A Quantitative Systems Approach Reveals Dynamic Control of tRNA Modifications during Cellular Stress

Decades of study have revealed more than 100 ribonucleoside structures incorporated as post-transcriptional modifications mainly in tRNA and rRNA, yet the larger functional dynamics of this conserved system are unclear. To this end, we developed a highly precise mass spectrometric method to quantify tRNA modifications in Saccharomyces cerevisiae. Our approach revealed several novel biosynthetic pathways for RNA modifications and led to the discovery of signature changes in the spectrum of tRNA modifications in the damage response to mechanistically different toxicants. This is illustrated with the RNA modifications Cm, m[superscript 5]C, and m[superscript 2][subscript 2]G, which increase following hydrogen peroxide exposure but decrease or are unaffected by exposure to methylmethane sulfonate, arsenite, and hypochlorite. Cytotoxic hypersensitivity to hydrogen peroxide is conferred by loss of enzymes catalyzing the formation of Cm, m[superscript 5]C, and m[superscript 2][subscript 2]G, which demonstrates that tRNA modifications are critical features of the cellular stress response. The results of our study support a general model of dynamic control of tRNA modifications in cellular response pathways and add to the growing repertoire of mechanisms controlling translational responses in cells.National Institute of Environmental Health Sciences (ES002109)National Institute of Environmental Health Sciences (ES017010)National Institute of Environmental Health Sciences (ES015037)National Cancer Institute (U.S.) (CA026731)National Center for Research Resources (U.S.) (RR023783)Singapore-MIT Alliance for Research and Technolog

A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for <it>Saccharomyces cerevisiae </it>to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid <it>S. cerevisiae </it>gene deletion strains (the entire set of nonessential genes for this organism) to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO₄).</p> <p>Results</p> <p>We have identified 149 genes whose gene deletion causes sensitivity to NiSO₄and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of <it>S. cerevisiae </it>to NiSO₄. Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO₄include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells.</p> <p>Conclusion</p> <p>We have undertaken a whole genome approach in order to further understand the mechanism(s) regulating the cell's toxicity to nickel compounds. We have used computational methods to integrate the data and generate global models of the yeast's cellular response to NiSO₄. The results of our study shed light on molecular pathways associated with the cellular response of eukaryotic cells to nickel compounds and provide potential implications for further understanding the toxic effects of nickel compounds to human cells.</p