Translational response to mitochondrial stresses is orchestrated by tRNA modifications.

Mitochondrial stress and dysfunction play important roles in many pathologies. However, how cells respond to mitochondrial stress is not fully understood. Here, we examined the translational response to electron transport chain (ETC) inhibition and arsenite induced mitochondrial stresses. Our analysis revealed that during mitochondrial stress, tRNA modifications (namely f5C, hm5C, queuosine and its derivatives, and mcm5U) dynamically change to fine tune codon decoding, usage, and optimality. These changes in codon optimality drive the translation of many pathways and gene sets, such as the ATF4 pathway and selenoproteins, involved in the cellular response to mitochondrial stress. We further examined several of these modifications using targeted approaches. ALKBH1 knockout (KO) abrogated f5C and hm5C levels and led to mitochondrial dysfunction, reduced proliferation, and impacted mRNA translation rates. Our analysis revealed that tRNA queuosine (tRNA-Q) is a master regulator of the mitochondrial stress response. KO of QTRT1 or QTRT2, the enzymes responsible for tRNA-Q synthesis, led to mitochondrial dysfunction, translational dysregulation, and metabolic alterations in mitochondria-related pathways, without altering cellular proliferation. In addition, our analysis revealed that tRNA-Q loss led to a domino effect on various tRNA modifications. Some of these changes could be explained by metabolic profiling. Our analysis also revealed that utilizing serum deprivation or alteration with Queuine supplementation to study tRNA-Q or stress response can introduce various confounding factors by altering many other tRNA modifications. In summary, our data show that tRNA modifications are master regulators of the mitochondrial stress response by driving changes in codon decoding.

DNA Modifications Enabling Proximity Biotinylation.

Advances in peroxidase and biotin ligase-mediated signal amplification have enabled high-resolution subcellular mapping of endogenous RNA localization and protein-protein interactions. Application of these technologies has been limited to RNA and proteins because of the reactive groups required for biotinylation in each context. Here we report several novel methods for proximity biotinylation of exogenous oligodeoxyribonucleotides by application of well-established and convenient enzymatic tools. We describe approaches using simple and efficient conjugation chemistries to modify deoxyribonucleotides with "antennae" that react with phenoxy radicals or biotinoyl-5'-adenylate. In addition, we report chemical details of a previously undescribed adduct between tryptophan and a phenoxy radical group. These developments have potential application in the selection of exogenous nucleic acids capable of unaided entry into living cells.

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12.

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.

Codon Usage and mRNA Stability are Translational Determinants of Cellular Response to Canonical Ferroptosis Inducers.

Ferroptosis is a non-apoptotic cell death mechanism characterized by the generation of lipid peroxides. While many effectors in the ferroptosis pathway have been mapped, its epitranscriptional regulation is not yet fully understood. Ferroptosis can be induced via system xCT inhibition (Class I) or GPX4 inhibition (Class II). Previous works have revealed important differences in cellular response to different ferroptosis inducers. Importantly, blocking mRNA transcription or translation appears to protect cells against Class I ferroptosis inducing agents but not Class II. In this work, we examined the impact of blocking transcription (via Actinomycin D) or translation (via Cycloheximide) on Erastin (Class I) or RSL3 (Class II) induced ferroptosis. Blocking transcription or translation protected cells against Erastin but was detrimental against RSL3. Cycloheximide led to increased levels of GSH alone or when co-treated with Erastin via the activation of the reverse transsulfuration pathway. RNA sequencing analysis revealed early activation of a strong alternative splice program before observed changes in transcription. mRNA stability analysis revealed divergent mRNA stability changes in cellular response to Erastin or RSL3. Importantly, codon optimality biases were drastically different in either condition. Our data also implicated translation repression and rate as an important determinant of the cellular response to ferroptosis inducers. Given that mRNA stability and codon usage can be influenced via the tRNA epitranscriptome, we evaluated the role of a tRNA modifying enzyme in ferroptosis stress response. Alkbh1, a tRNA demethylase, led to translation repression and increased the resistance to Erastin but made cells more sensitive to RSL3.

Strategies to Avoid Artifacts in Mass Spectrometry-Based Epitranscriptome Analyses.

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2'-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.

Unraveling Reversible DNA Cross-Links with a Biological Machine.

The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of alkylation and cross-linking. This reversibility also expands the possible consequences that can be envisioned when confronted by DNA repair processes and biological machines. To begin testing the response to such an encounter, quinone methide-based modification of DNA has now been challenged with a helicase (T7 bacteriophage gene protein four, T7gp4) that promotes 5' to 3' translocation and unwinding. This model protein was selected based on its widespread application, well characterized mechanism and detailed structural information. Little over one-half of the cross-linking generated by a bisfunctional quinone methide remained stable to T7gp4 and did not suppress its activity. The helicase likely avoids the topological block generated by this fraction of cross-linking by its ability to shift from single- to double-stranded translocation. The remaining fraction of cross-linking was destroyed during T7gp4 catalysis. Thus, this helicase is chemically competent to promote release of the quinone methide from DNA. The ability of T7gp4 to act as a Brownian ratchet for unwinding DNA may block recapture of the QM intermediate by DNA during its transient release from a donor strand. Most surprisingly, T7gp4 releases the quinone methide from both the translocating strand that passes through its central channel and the excluded strand that was typically unaffected by other lesions. The ability of T7gp4 to reverse the cross-link formed by the quinone methide does not extend to that formed irreversibly by the nitrogen mustard mechlorethamine.

Directing Quinone Methide-Dependent Alkylation and Cross-Linking of Nucleic Acids with Quaternary Amines.

Polyamine and polyammonium ion conjugates are often used to direct reagents to nucleic acids based on their strong electrostatic attraction to the phosphoribose backbone. Such nonspecific interactions do not typically alter the specificity of the attached reagent, but polyammonium ions dramatically redirected the specificity of a series of quinone methide precursors. Replacement of a relatively nonspecific intercalator based on acridine with a series of polyammonium ions resulted in a surprising change of DNA products. Piperidine stable adducts were generated in duplex DNA that lacked the ability to support a dynamic cross-linking observed previously with acridine conjugates. Minor reaction at guanine N7, the site of reversible reaction, was retained by a monofunctional quinone methide-polyammonium ion conjugate, but a bisfunctional analogue designed for tandem quinone methide formation modified guanine N7 in only single-stranded DNA. The resulting intrastrand cross-links were sufficiently dynamic to rearrange to interstrand cross-links. However, no further transfer of adducts was observed in duplex DNA. An alternative design that spatially and temporally decoupled the two quinone methide equivalents neither restored the dynamic reaction nor cross-linked DNA efficiently. While di- and triammonium ion conjugates successfully enhanced the yields of cross-linking by a bisquinone methide relative to a monoammonium equivalent, alternative ligands will be necessary to facilitate the migration of cross-linking and its potential application to disrupt DNA repair.

Effect of Nucleosome Assembly on Alkylation by a Dynamic Electrophile.

Quinone methides are reactive electrophiles that are generated during metabolism of various drugs, natural products, and food additives. Their chemical properties and cellular effects have been described previously, and now their response to packaging DNA in a nucleosome core is described. A model bisquinone methide precursor (bisQMP) was selected based on its ability to form reversible adducts with guanine N7 that allow for their redistribution and transfer after quinone methide regeneration. Assembly of Widom's 601 DNA with the histone octamer of H2A, H2B, H3, and H4 from Xenopus laevis significantly suppressed alkylation of the DNA. This result is a function of DNA packaging since addition of the octamer without nucleosome reconstitution only mildly protected DNA from alkylation. The lack of competition between nucleophiles of DNA and the histones was consistent with the limited number of adducts formed by the histones as detected by tryptic digestion and ultraperformance liquid chromatography-mass spectrometry. Only three peptide adducts were observed after reaction with a monofunctional analogue of bisQMP, and only two peptide adducts were observed after reaction with bisQMP. Histone reaction was also suppressed when reconstituted into the nucleosome core particle. However, bisQMP was capable of cross-linking the DNA and histones in moderate yields (∼20%) that exceeded expectations derived from reaction of cisplatin, nitrogen mustards, and diepoxybutane. The core histones also demonstrated a protective function against dynamic alkylation by trapping the reactive quinone methide after its spontaneous regeneration from DNA adducts.