Oligomerization and a distinct tRNA-binding loop are important regulators of human arginyl-transferase function.

The arginyl-transferase ATE1 is a tRNA-dependent enzyme that covalently attaches an arginine molecule to a protein substrate. Conserved from yeast to humans, ATE1 deficiency in mice correlates with defects in cardiovascular development and angiogenesis and results in embryonic lethality, while conditional knockouts exhibit reproductive, developmental, and neurological deficiencies. Despite the recent revelation of the tRNA binding mechanism and the catalytic cycle of yeast ATE1, the structure-function relationship of ATE1 in higher organisms is not well understood. In this study, we present the three-dimensional structure of human ATE1 in an apo-state and in complex with its tRNA cofactor and a peptide substrate. In contrast to its yeast counterpart, human ATE1 forms a symmetric homodimer, which dissociates upon binding of a substrate. Furthermore, human ATE1 includes a unique and extended loop that wraps around tRNAArg, creating extensive contacts with the T-arm of the tRNA cofactor. Substituting key residues identified in the substrate binding site of ATE1 abolishes enzymatic activity and results in the accumulation of ATE1 substrates in cells.

Copper catalyzed cycloaddition for the synthesis of non isomerisable 2' and 3'-regioisomers of arg-tRNAarg.

In this report, non-isomerisable analogs of arginine tRNA (Arg-triazole-tRNA) have been synthesized as tools to study tRNA-dependent aminoacyl-transferases. The synthesis involves the incorporation of 1,4 substituted-1,2,3 triazole ring to mimic the ester bond that connects the amino acid to the terminal adenosine in the natural substrate. The synthetic procedure includes (i) a coupling between 2'- or 3'-azido-adenosine derivatives and a cytidine phosphoramidite to access dinucleotide molecules, (ii) Cu-catalyzed cycloaddition reactions between 2'- or 3'-azido dinucleotide in the presence of an alkyne molecule mimicking the arginine, providing the corresponding Arg-triazole-dinucleotides, (iii) enzymatic phosphorylation of the 5'-end extremity of the Arg-triazole-dinucleotides with a polynucleotide kinase, and (iv) enzymatic ligation of the 5'-phosphorylated dinucleotides with a 23-nt RNA micro helix that mimics the acceptor arm of arg-tRNA or with a full tRNAarg. Characterization of nucleoside and nucleotide compounds involved MS spectrometry, 1H, 13C and 31P NMR analysis. This strategy allows to obtain the pair of the two stable regioisomers of arg-tRNA analogs (2' and 3') which are instrumental to explore the regiospecificity of arginyl transferases enzyme. In our study, a first binding assay of the arg-tRNA micro helix with the Arginyl-tRNA-protein transferase 1 (ATE1) was performed by gel shift assays.

Reporter Gene-Based qRT-PCR Assay for Rho-Dependent Termination In Vivo.

In bacteria, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we could not identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene argX, (coding tRNAarg) from Brevibacterium albidum, and we transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE, galT, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. Since the quantitative real-time PCR (qRT-PCR) method detects the steady state following mRNA synthesis and degradation, we observed that tRNAarg is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg can directly reflect the mRNA synthesis. Using this approach, we were able to effectively assay the RDTs and Rho-independent termination (RIT) in the gal operon by quantifying the relative amount of tRNAarg using qRT-PCR analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. The resultant RIT% at the end of the gal operon is 33%. Our findings demonstrate that combining tRNAarg with qRT-PCR can directly measure RIT, RDT, or any other signal that attenuates transcription efficiencies in vivo, making it a useful tool for gene expression research.

yaaJ, the tRNA-Specific Adenosine Deaminase, Is Dispensable in Bacillus subtilis.

Post-transcriptional modifications of tRNA are crucial for their core function. The inosine (I; 6-deaminated adenosine) at the first position in the anticodon of tRNAArg(ICG) modulates the decoding capability and is generally considered essential for reading CGU, CGC, and CGA codons in eubacteria. We report here that the Bacillus subtilis yaaJ gene encodes tRNA-specific adenosine deaminase and is non-essential for viability. A ß-galactosidase reporter assay revealed that the translational activity of CGN codons was not impaired in the yaaJ-deletion mutant. Furthermore, tRNAArg(CCG) responsible for decoding the CGG codon was dispensable, even in the presence or absence of yaaJ. These results strongly suggest that tRNAArg with either the anticodon ICG or ACG has an intrinsic ability to recognize all four CGN codons, providing a fundamental concept of non-canonical wobbling mediated by adenosine and inosine nucleotides in the anticodon. This is the first example of the four-way wobbling by inosine nucleotide in bacterial cells. On the other hand, the absence of inosine modification induced +1 frameshifting, especially at the CGA codon. Additionally, the yaaJ deletion affected growth and competency. Therefore, the inosine modification is beneficial for translational fidelity and proper growth-phase control, and that is why yaaJ has been actually conserved in B. subtilis.

[Correlation of mitochondrial tRNA variants with coronary heart disease in a Chinese pedigree].

OBJECTIVE: To explore the correlation of mitochondrial DNA (mtDNA) variants and coronary heart disease (CHD) in a Chinese pedigree and the possible molecular mechanisms. METHODS: A Chinese pedigree featuring matrilineal inheritance of CHD who visited Hangzhou First People's Hospital in May 2022 was selected as the study subject. Clinical data of the proband and her affected relatives was collected. By sequencing the mtDNA of the proband and her pedigree members, candidate variants were identified through comparison with wild type mitochondrial genes. Conservative analysis among various species was conducted, and bioinformatics software was used to predict the impact of variants on the secondary structure of tRNA. Real-time PCR was carried out to determine the copy number of mtDNA, and a transmitochondrial cell line was established for analyzing the mitochondrial functions, including membrane potential and ATP level. RESULTS: This pedigree had contained thirty-two members from four generations. Among ten maternal members, four had CHD, which yielded a penetrance rate of 40%. Sequence analysis of proband and her matrilineal relatives revealed the presence of a novel m.4420A>T variant and a m.10463T>C variant, both of which were highly conserved among various species. Structurally, the m.4420A>T variant had occurred at position 22 in the D-arm of tRNAMet, which disrupted the 13T-22A base-pairing, while the m.10463T>C variant was located at position 67 in the acceptor arm of tRNAArg, a position critical for steady-state level of the tRNA. Functional analysis revealed that patients with the m.4420A>T and m.10463T>C variants exhibited much fewer copy number of mtDNA and lower mitochondrial membrane potential (MMP) and ATP contents (P < 0.05), which were decreased by approximately 50.47%, 39.6% and 47.4%, respectively. CONCLUSION: Mitochondrial tRNAMet 4420A>T and tRNAArg 10463T>C variants may underlay the maternally transmitted CHD in this pedigree, which had shown variation in mtDNA homogeneity, age of onset, clinical phenotype and other differences, suggesting that nuclear genes, environmental factors and mitochondrial genetic background have certain influence on the pathogenesis of CHD.

Preparation of tRNAArg for Arginylation Assay by In Vitro Transcription.

This chapter describes the preparation of tRNAArg by in vitro transcription. tRNA produced by this method can be efficiently utilized for in vitro arginylation assays, following aminoacylation with Arg-tRNA synthetase, either directly during the arginylation reaction or separately to produce the purified preparation of Arg-tRNAArg. tRNA charging is described in other chapters of this book.

Preparation of an Enriched tRNAArg Fraction for Arginylation by Expression in E. coli.

The method described here provides a fast and efficient way to obtain an enriched preparation of tRNA of interest, which is also posttranscriptionally modified by the intracellular machinery of the host cells, E. coli. While this preparation also contains a mixture of total E. coli tRNA, the enriched tRNA of interest is obtained in high yields (milligram) and is highly efficient for biochemical assays in vitro. It is routinely used in our lab for arginylation.

Enzymatic Aminoacylation of tRNAArg Using Recombinant Arg-tRNA Synthetase.

This chapter describes the preparation of pre-charged Arg-tRNA that can be used in arginylation reaction. While in a typical arginylation reaction arginyl-tRNA synthetase (RARS) is normally included as a component of the reaction and continually charges tRNA during arginylation, it is sometimes necessary to separate the charging and the arginylation step, in order to perform each reaction under controlled conditions, e.g., for measuring the kinetics or determining the effect of different compounds and chemicals on the reaction. In such cases, tRNAArg can be pre-charged with Arg and purified away from the RARS enzyme prior to arginylation.

Synthesis of Stably Charged Arg-tRNAArg for Structural Analysis.

Posttranslational protein arginylation catalyzed by arginyl transferases is a mechanism to regulate multiple physiological processes. This protein arginylation reaction uses a charged Arg-tRNAArg as the donor of arginine (Arg). The inherent instability of the ester linkage of the arginyl group to the tRNA, which is sensitive to hydrolysis at the physiological pH, makes it difficult to obtain structural information on how the arginyl transfer reaction is catalyzed. Here, we describe a methodology to synthesize stably charged Arg-tRNAArg that would facilitate structural analysis. In the stably charged Arg-tRNAArg, the ester linkage is replaced with an amide linkage, which is resistant to hydrolysis even at alkaline pH.

The CGA codon decoding through tRNAArg (ICG) supply governed by Tad2/Tad3 in Saccharomyces cerevisiae.

The CGA codon is a rare codon in Saccharomyces cerevisiae and is known to be inefficiently decoded by wobble pairing with Arg-tRNA(ICG). The tRNAArg (ICG) is post-transcriptionally edited from tRNAArg (ACG) by the anticodon first adenosine deamination enzyme Tad2/Tad3 complex. Experimental consecutive CGA codons cause ribosome stalling to result in the reduction of the encoding protein product. In this study, the additional supply of tRNAArg (ACG) genes that produce decoding Arg-tRNA(ICG) promoted the product level from the CGA12-luc reporter, revealing that the product reduction is essentially due to inefficient decoding and deficiency in the tRNA supply. The mature tRNAArg (ICG) and the precursor tRNAArg (ACG) ratios examined for cellular tRNA fraction revealed that the tRNAArg (ICG) ratio is maintained at less than 30% and is responsive to the Tad2/Tad3 expression level.

A standalone editing protein deacylates mischarged canavanyl-tRNAArg to prevent canavanine incorporation into proteins.

Error-free translation of the genetic code into proteins is vitally important for all organisms. Therefore, it is crucial that the correct amino acids are loaded onto their corresponding tRNAs. This process is highly challenging when aminoacyl-tRNA-synthetases encounter structural analogues to the native substrate like the arginine antimetabolite canavanine. To circumvent deleterious incorporation due to tRNA mischarging, editing mechanisms have evolved. However, only for half of the tRNA synthetases, editing activity is known and only few specific standalone editing proteins have been described. Understanding the diverse mechanisms resulting in error-free protein synthesis is of great importance. Here, we report the discovery of a protein that is upregulated upon canavanine stimulation in bacteria that live associated with canavanine-producing plants. We demonstrate that it acts as standalone editing protein specifically deacylating canavanylated tRNAArg. We therefore propose canavanyl-tRNAArgdeacylase (CtdA) as systematic name. Knockout strains show severe growth defects in canavanine-containing media and incorporate high amounts of canavanine into the proteome. CtdA is frequently found under control of guanidine riboswitches, revealing a functional connection of canavanine and guanidine metabolisms. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Error-free translation is one of the most vital processes in all living organisms, but can be substantially challenged by compounds that mimic amino acids. Canavanine, or 5-oxa-arginine, is used as an antimetabolite by higher plants that is toxic due to its incorporation into proteins. We report the discovery of a standalone editing protein specifically deacylating canavanylated tRNAArg that enables the legume rhizosphere inhabitant Pseudomonas canavaninivorans to prevent canavanine mis-incorporation into its proteome. Our results are the first to show editing activity towards mischarged tRNAArg and add to the puzzle of how faithful translation is ensured in nature.

Correlation of mitochondrial tRNA variants with coronary heart disease in a Chinese pedigree / 中华医学遗传学杂志

OBJECTIVE@#To explore the correlation of mitochondrial DNA (mtDNA) variants and coronary heart disease (CHD) in a Chinese pedigree and the possible molecular mechanisms.@*METHODS@#A Chinese pedigree featuring matrilineal inheritance of CHD who visited Hangzhou First People's Hospital in May 2022 was selected as the study subject. Clinical data of the proband and her affected relatives was collected. By sequencing the mtDNA of the proband and her pedigree members, candidate variants were identified through comparison with wild type mitochondrial genes. Conservative analysis among various species was conducted, and bioinformatics software was used to predict the impact of variants on the secondary structure of tRNA. Real-time PCR was carried out to determine the copy number of mtDNA, and a transmitochondrial cell line was established for analyzing the mitochondrial functions, including membrane potential and ATP level.@*RESULTS@#This pedigree had contained thirty-two members from four generations. Among ten maternal members, four had CHD, which yielded a penetrance rate of 40%. Sequence analysis of proband and her matrilineal relatives revealed the presence of a novel m.4420A>T variant and a m.10463T>C variant, both of which were highly conserved among various species. Structurally, the m.4420A>T variant had occurred at position 22 in the D-arm of tRNAMet, which disrupted the 13T-22A base-pairing, while the m.10463T>C variant was located at position 67 in the acceptor arm of tRNAArg, a position critical for steady-state level of the tRNA. Functional analysis revealed that patients with the m.4420A>T and m.10463T>C variants exhibited much fewer copy number of mtDNA and lower mitochondrial membrane potential (MMP) and ATP contents (P < 0.05), which were decreased by approximately 50.47%, 39.6% and 47.4%, respectively.@*CONCLUSION@#Mitochondrial tRNAMet 4420A>T and tRNAArg 10463T>C variants may underlay the maternally transmitted CHD in this pedigree, which had shown variation in mtDNA homogeneity, age of onset, clinical phenotype and other differences, suggesting that nuclear genes, environmental factors and mitochondrial genetic background have certain influence on the pathogenesis of CHD.

Microevolution of CG23-I Hypervirulent Klebsiella pneumoniae during Recurrent Infections in a Single Patient.

CG23-I lineage constitutes the majority of hypervirulent Klebsiella pneumoniae. A diabetic patient suffered six episodes of infections caused by CG23-I K. pneumoniae. A total of nine isolates were collected in 2020. We performed whole-genome sequencing to elucidate the within-patient evolution of CG23-I K. pneumoniae. The maximum pairwise difference among the nine longitudinally collected isolates was five single nucleotide polymorphisms. One of the mutations was at the Asp87 position of GyrA. Four indels were identified, including an initiator tRNAfMet duplication, a tRNAArg deletion, a 7-bp insertion, and a 22-bp deletion. All 9 isolates had the genomic features of CG23-I K. pneumoniae, a chromosome-borne ICEKp10, and a large virulence plasmid. The carriage of a complete set of genes for the biosynthesis of colibactin by ICEKp10 gave the nine isolates an ability to cause DNA damage to RAW264.7 cells. Compared with the initial isolate, the last isolate with an additional copy of initiator tRNAfMet grew faster in a nutrient-limiting condition and exhibited enhanced virulence in BALB/c mice. Collectively, we characterized the within-patient microevolution of CG23-I K. pneumoniae through an in-depth comparison of genome sequences. Using the in vitro experiments and mouse models, we also demonstrated that these genomic alterations endowed the isolates with advantages to pass through in vivo selection. IMPORTANCE CG23-I is a significant lineage of hypervirulent Klebsiella pneumoniae. This study characterizes the within-patient microevolution of CG23-I K. pneumoniae. Selective pressures from continuous use of antibiotics favored point mutations contributing to bacterial resistance to antibiotics. The duplication of an initiator tRNAfMet gene helped CG23-I K. pneumoniae proliferate to reach a maximal population size during infections. For longer persistence inside a human host, the large virulence plasmid evolved with more flexible control of replication through duplication of the iteron-1 region. With the genomic alterations, the last isolate had a growth advantage over the initial isolate and exhibited enhanced virulence in BALB/c mice. This study gives us a deeper understanding of the genome evolution during the within-patient pathoadaptation of CG23-I K. pneumoniae.

Functional Interplay between Arginyl-tRNA Synthetases and Arginyltransferase.

Protein arginylation, mediated by arginyltransferase ATE1, is a post-translational modification of emerging biological importance that consists of transfer of the amino acid Arg to protein and peptide substrates. ATE1 utilizes charged tRNAArg as the donor of the arginyl group, which depends on the activity of Arg-tRNA synthetases (RARS) and is also utilized in translation. The mechanisms that regulate the functional balance among ATE1, RARS and translation are unknown. Here, we addressed the question of how these two enzymes can partition Arg-tRNAArg to functionally distinct pathways using an intracellular arginylation sensor in cell lines with overexpression or deletion of ATE1 and RARS isoforms. We found that arginylation levels depend on the physiological state of the cells but are not directly affected by translation activity or the availability of RARS isoforms. However, displacement of RARS from the multi-synthetase complex leads to an increase in intracellular arginylation independently of RARS enzymatic activity. This effect is accompanied by ATE1's redistribution into the cytosol. Our results provide the first comprehensive analysis of the interdependence among translation, arginyl-tRNA synthesis and arginylation.

Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates.

N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNAArg as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNAArg, and a 3'-proximal segment of the tRNAArg moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe3+-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.

Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins.

Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.

Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems.

CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.

Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex.

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNAArg and tRNAGln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.

Substrate recognition mechanism of tRNA-targeting ribonuclease, colicin D, and an insight into tRNA cleavage-mediated translation impairment.

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNAArg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNAArgs; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNAArgs from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNAArgICG, the most abundant species of E. coli tRNAArgs, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNAArgICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNAArgICG dominant-negatively impairs translation in vitro. Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNAArgICG, which is consistent with our structural model. Finally, elevation of cleaved tRNAArgICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNAArgICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops.Abbreviations: mnm5U: 5-methylaminomethyluridine; mcm5s2U: 5-methoxycarbonylmethyl-2-thiouridine.

Expression of the Neuronal tRNA n-Tr20 Regulates Synaptic Transmission and Seizure Susceptibility.

The mammalian genome has hundreds of nuclear-encoded tRNAs, but the contribution of individual tRNA genes to cellular and organismal function remains unknown. Here, we demonstrate that mutations in a neuronally enriched arginine tRNA, n-Tr20, increased seizure threshold and altered synaptic transmission. n-Tr20 expression also modulated seizures caused by an epilepsy-linked mutation in Gabrg2, a gene encoding a GABAA receptor subunit. Loss of n-Tr20 altered translation initiation by activating the integrated stress response and suppressing mTOR signaling, the latter of which may contribute to altered neurotransmission in mutant mice. Deletion of a highly expressed isoleucine tRNA similarly altered these signaling pathways in the brain, suggesting that regulation of translation initiation is a conserved response to tRNA loss. Our data indicate that loss of a single member of a tRNA family results in multiple cellular phenotypes, highlighting the disease-causing potential of tRNA mutations.