DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3'-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3'-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.

Here we present the Nick-seq platform for quantitative mapping of DNA modifications and damage at single-nucleotide resolution across genomes. Pre-existing breaks are blocked and DNA structures converted to strand-breaks for 3’-extension by nick-translation to produce nuclease-resistant oligonucleotides, and 3’-capture by terminal transferase tailing. Libraries from both products are subjected to next-generation sequencing. Nick-seq is a generally applicable method illustrated with quantitative profiling of single-strand-breaks, phosphorothioate modifications, and DNA oxidation.

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.

Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra‐erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up‐regulation, and asynchronous changes in six modifications. Quantitative analysis of 2,100 proteins across the IDC revealed that up‐ and down‐regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage‐specific proteins by enhancing translation efficiency of codon‐biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites. Using a LC‐MS/MS based method, we characterize tRNA modifications in P. falciparum. Here we present a model integrating these epitranscriptomic changes and codon‐biased translation based on a quantitative systems‐level analysis of the genome, transcriptome and the proteome across the parasite's asexual lifecycle. Using a LC‐MS/MS based method, we characterize tRNA modifications in P. falciparum. Here we present a model integrating these epitranscriptomic changes and codon‐biased translation based on a quantitative systems‐level analysis of the genome, transcriptome and the proteome across the parasite's asexual lifecycle.

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.

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. This 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.

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.

SummaryProkaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction–modification (R–M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R–M systems, highly partial PT modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT‐dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB–E, while restriction involves additional three genes dptF–H. Here, we performed a series of studies to characterize the PT‐dependent restriction, and found that it presented several features distinct with traditional R–M systems. The presence of restriction genes in a PT‐deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of > 600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair‐related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, overexpression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT‐dependent DNA cleavage by restriction enzymes in the face of partial PT modification.

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 crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. 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 via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.

The mTOR pathway is a conserved master regulator of translational activity that influences the fate of industrially relevant CHO cell cultures, yet its molecular mechanisms remain unclear. Interestingly, rapamycin specific inhibition of the mTOR pathway in CHO cells was found to down-regulate the small nucleolar RNA U19 (snoRNA U19) by 2-fold via translatome profiling. snoRNA U19 guides the two most conserved pseudouridylation modifications on 28S ribosomal RNA (rRNA) that are important for the biogenesis and proper function of ribosomes. In order to further understand the role of snoRNA U19 as a potential player in the mTOR pathway, we measured 28S rRNA pseudouridylation upon rapamycin treatments and/or snoRNA U19 overexpression conditions, thereby characterizing the subsequent effects on ribosome efficiency and global translation by polysome profiling. We showed that 28S rRNA pseudouridylation was increased by rapamycin treatment and/or overexpression of snoRNA U19, but only the latter condition improved ribosome efficiency toward higher global translation, thus implying that the mTOR pathway induces pseudouridylation at different sites along the 28S rRNA possibly with either positive or negative effects on the cellular phenotype. This discovery of snoRNA U19 as a new downstream effector of the mTOR pathway suggests that cell engineering of snoRNAs can be used to regulate translation and improve cellular growth in CHO cell cultures in the future.

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.

At high ambient temperature, plants display dramatic stem elongation in an adaptive response to heat. This response is mediated by elevated levels of the phytohormone auxin and requires auxin biosynthesis, signaling, and transport pathways. The mechanisms by which higher temperature results in greater auxin accumulation are unknown, however. A basic helix-loop-helix transcription factor, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), is also required for hypocotyl elongation in response to high temperature. PIF4 also acts redundantly with its homolog, PIF5, to regulate diurnal growth rhythms and elongation responses to the threat of vegetative shade. PIF4 activity is reportedly limited in part by binding to both the basic helix-loop-helix protein LONG HYPOCOTYL IN FAR RED 1 and the DELLA family of growth-repressing proteins. Despite the importance of PIF4 in integrating multiple environmental signals, the mechanisms by which PIF4 controls growth are unknown. Here we demonstrate that PIF4 regulates levels of auxin and the expression of key auxin biosynthesis genes at high temperature. We also identify a family of SMALL AUXIN UP RNA (SAUR) genes that are expressed at high temperature in a PIF4-dependent manner and promote elongation growth. Taken together, our results demonstrate direct molecular links among PIF4, auxin, and elongation growth at high temperature.