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Synthetic phosphorothioate (PT) internucleotide linkages, in which a nonbridging oxygen is replaced by a sulphur atom, share similar physical and chemical properties with phosphodiesters but confer enhanced nuclease tolerance on DNA/RNA, making PTs a valuable biochemical and pharmacological tool. Interestingly, PT modification was recently found to occur naturally in bacteria in a sequence-selective and RP configuration-specific manner. This oxygen-sulphur swap is catalysed by the gene products of dndABCDE, which constitute a defence barrier with DndFGH in some bacterial strains that can distinguish and attack non-PT-modified foreign DNA, resembling DNA methylation-based restriction-modification (R-M) systems. Despite their similar defensive mechanisms, PT- and methylation-based R-M systems have evolved to target different consensus contexts in the host cell because when they share the same recognition sequences, the protective function of each can be impeded. The redox and nucleophilic properties of PT sulphur render PT modification a versatile player in the maintenance of cellular redox homeostasis, epigenetic regulation and environmental fitness. The widespread presence of dnd systems is considered a consequence of extensive horizontal gene transfer, whereas the lability of PT during oxidative stress and the susceptibility of PT to PT-dependent endonucleases provide possible explanations for the ubiquitous but sporadic distribution of PT modification in the bacterial world.

Chemical modifications of the nucleosides that comprise transfer RNAs are diverse. However, the structure, location and extent of modifications have been systematically charted in very few organisms. Here, we describe an approach in which rapid prediction of modified sites through reverse transcription-derived signatures in high-throughput transfer RNA-sequencing (tRNA-seq) data is coupled with identification of tRNA modifications through RNA mass spectrometry. Comparative tRNA-seq enabled prediction of several Vibrio cholerae modifications that are absent from Escherichia coli and also revealed the effects of various environmental conditions on V. cholerae tRNA modification. Through RNA mass spectrometric analyses, we showed that two of the V. cholerae- specific reverse transcription signatures reflected the presence of a new modification (acetylated acp 3 U (acacp 3 U)), while the other results from C-to-Ψ RNA editing, a process not described before. These findings demonstrate the utility of this approach for rapid surveillance of tRNA modification profiles and environmental control of tRNA modification. A combination of tRNA sequencing and mass spectrometry was developed to profile tRNA modification enabling identification of a new modification acacp 3 U and the presence of cytosine-to-pseudouracil RNA editing in Vibrio cholerae .

Cells adapt to stress by altering gene expression at multiple levels. Here, we propose a new mechanism regulating stress-dependent gene expression at the level of translation, with coordinated interplay between the tRNA epitranscriptome and biased codon usage in families of stress-response genes. In this model, auxiliary genetic information contained in synonymous codon usage enables regulation of codon-biased and functionally related transcripts by dynamic changes in the tRNA epitranscriptome. This model partly explains the association between synchronous stress-dependent epitranscriptomic marks and significant multi-codon usage skewing in families of translationally regulated transcripts. The model also predicts translational adaptation during viral infections.

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.

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(5)C, and m(2) (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(5)C, and m(2) (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.

Selective translation of survival proteins is an important facet of the cellular stress response. We recently demonstrated that this translational control involves a stress-specific reprogramming of modified ribonucleosides in tRNA. Here we report the discovery of a step-wise translational control mechanism responsible for survival following oxidative stress. In yeast exposed to hydrogen peroxide, there is a Trm4 methyltransferase-dependent increase in the proportion of tRNA Leu( C AA) containing m 5 C at the wobble position, which causes selective translation of mRNA from genes enriched in the TTG codon. Of these genes, oxidative stress increases protein expression from the TTG-enriched ribosomal protein gene RPL22A , but not its unenriched paralogue. Loss of either TRM4 or RPL22A confers hypersensitivity to oxidative stress. Proteomic analysis reveals that oxidative stress causes a significant translational bias towards proteins coded by TTG-enriched genes. These results point to stress-induced reprogramming of tRNA modifications and consequential reprogramming of ribosomes in translational control of cell survival. In response to stress, yeast cells selectively translate proteins that can enhance cell survival. In this study, the authors find that tRNA LEU(CAA) in yeast cells is modified in response to oxidative stress by a methyltransferase and this results in the selective translation of the mRNA for these genes.

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 cmo 5 U in tRNA Thr(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. Mycobacteria can adapt to the stress of human infection by entering a dormant state. Here the authors show that hypoxia-induced dormancy in M. bovis BCG involves the reprogramming of tRNA wobble modifications and copy numbers, coupled with biased use of synonymous codons in survival genes.

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.

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.

Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum , but few factors have been shown to play a direct role. We adapted a CRISPR‐based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 (“dCas9”) was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of Pf ISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of Pf ISWI identified several proteins enriched at the var gene promoter such as acetyl‐CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene‐associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin‐associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite. Synopsis CRISPR/dCas9‐based proteomics is used to purify specific DNA regulatory elements in their natural chromatin context and to identify novel chromatin factors associated with virulence genes in the human malaria parasite, Plasmodium falciparum . dCas9 immunoprecipitation and mass spectrometry identify proteins previously implicated in var gene biology in addition to novel factors, including a putative chromatin remodeler, ISWI. Proteomic analysis of ISWI reveals a new var gene‐associated complex comprising a putative MORC family protein and an ApiAP2 transcription factor. ISWI binds to promoter regions and plays a role in transcriptional activation of genes, including the active var gene in ring stage parasites. Graphical Abstract CRISPR/dCas9‐based proteomics is used to purify specific DNA regulatory elements in their natural chromatin context and to identify novel chromatin factors associated with virulence genes in the human malaria parasite, Plasmodium falciparum .