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

SBML Level 3: an extensible format for the exchange and reuse of biological models

Systems biology has experienced dramatic growth in the number, size, and complexity of computational models. To reproduce simulation results and reuse models, researchers must exchange unambiguous model descriptions. We review the latest edition of the Systems Biology Markup Language (SBML), a format designed for this purpose. A community of modelers and software authors developed SBML Level 3 over the past decade. Its modular form consists of a core suited to representing reaction-based models and packages that extend the core with features suited to other model types including constraint-based models, reaction-diffusion models, logical network models, and rule-based models. The format leverages two decades of SBML and a rich software ecosystem that transformed how systems biologists build and interact with models. More recently, the rise of multiscale models of whole cells and organs, and new data sources such as single-cell measurements and live imaging, has precipitated new ways of integrating data with models. We provide our perspectives on the challenges presented by these developments and how SBML Level 3 provides the foundation needed to support this evolution

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

Two self-splicing group I introns in the ribonucleotide reductase large subunit gene of Staphylococcus aureus phage Twort

We have recently described three group I introns inserted into a single gene, orf142, of the staphylococcal bacteriophage Twort and suggested the presence of at least two additional self-splicing introns in this phage genome. Here we report that two previously uncharacterized introns, 429 and 1087 nt in length, interrupt the Twort gene coding for the large subunit of ribonucleotide reductase (nrdE). Reverse transcription–polymerase chain reaction (RT–PCR) of RNA isolated from Staphylococcus aureus after phage infection indicates that the introns are removed from the primary transcript in vivo. Both nrdE introns show sequence similarity to the Twort orf142 introns I2 and I3, suggesting either a common origin of these introns or shuffling of intron structural elements. Intron 2 encodes a DNA endonuclease, I-TwoI, with similarity to homing endonucleases of the HNH family. Like I-HmuI and I-HmuII, intron-encoded HNH endonucleases in Bacillus subtilis phages SPO1 and SP82, I-TwoI nicks only one strand of its DNA recognition sequence. However, whereas I-HmuI and I-HmuII cleave the template strand in exon 2, I-TwoI cleaves the coding strand in exon 1. In each case, the 3′ OH created on the cut strand is positioned to prime DNA synthesis towards the intron, suggesting that this reaction contributes to the mechanism of intron homing. Both nrdE introns are inserted in highly conserved regions of the ribonucleotide reductase gene, next to codons for functionally important residues

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 similar to 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.ISSN:0027-8424ISSN:1091-649