Tryptophan codon-specific mistranslation, in the form of ribosomal frameshifting and tryptophan-to-phenylalanine (W > F) codon reassignments (substitutants), is induced in cancer cells by the limiting level of tryptophan imposed by anti-tumour immunity. While the oncogenic mitogen-activated protein kinase pathway drives frameshifting, whether substitutants are genetically regulated remains unknown. Here we screened for genes that control W > F substitutants following interferon-γ-mediated tryptophan shortage. This screen identified ADAR1, an enzyme that converts adenosine to inosine in double-stranded RNA molecules, and FTSJ1, an enzyme that 2'-O-methylates the anticodon region of several tRNAs. We demonstrate that ADAR1 sustains expression of key players in the ribosome quality control pathway, which in turn is essential for mistranslation events. FTSJ1, in contrast, specifically drives W > F mistranslation by methylation of tRNA[Trp] to promote its binding to WARS1 loaded with phenylalanine instead of tryptophan. As ADAR1 and FTSJ1 levels are elevated in many cancer types, we propose that cancer cells deploy global and specific mechanisms to promote mistranslation in response to anti-tumour immunity.

A central dogma of molecular biology is the "speed-accuracy trade-off," where ribosomes must slow down to ensure accurate protein synthesis. In mycobacteria, a high basal level of mistranslation at glutamine and asparagine codons, caused by an indirect tRNA aminoacylation pathway, promotes tolerance to the antibiotic rifampicin. While pharmacologically increasing translational fidelity is a promising strategy to combat antibiotic tolerance, the underlying mechanisms remain poorly understood. Here, we screened 9,000 synthetic compounds and identified benzo[d]isoxazole-4,7-diones as a novel chemical class that reduces mycobacterial mistranslation. Medicinal chemistry optimization yielded a lead compound, 9787, with superior potency in decreasing mistranslation and reversing rifampicin tolerance. Using competitive chemical proteomics, we identified the 30S ribosomal protein S5 (RpS5) as the specific cellular target. Remarkably, compound 9787 enhances translational fidelity at concentrations that do not measurably impact the overall rate of protein synthesis. Our findings challenge the universality of the speed-accuracy trade-off, demonstrating that fidelity can be improved independently of translation speed. This work reveals that the ribosomal small subunit is a druggable target for modulating translational quality control, and introduces a new strategy for combating antibiotic-tolerant bacteria without the associated fitness cost of slowed translation.IMPORTANCEA fundamental principle in molecular biology holds that ribosomes face a trade-off between translation speed and accuracy: going faster means making more errors, while maintaining high fidelity requires slowing down. This study challenges that paradigm by identifying a small molecule that increases translational accuracy in mycobacteria without affecting the rate of protein synthesis. The compound targets ribosomal protein S5 and specifically reduces errors arising from physiologically mischarged tRNAs-a quality control problem distinct from the well-studied codon-anticodon mismatches. This form of mistranslation contributes to antibiotic tolerance in tuberculosis, making it a potential therapeutic target. Our findings reveal unexpected flexibility in how ribosomes maintain translation quality and suggest that pharmacologically increasing fidelity without the fitness cost of slowed protein synthesis may be an attractive strategy for combating antibiotic-tolerant bacteria.

Protein errors (amino acid misincorporations) are associated to protein misfolding and misfunction, and lower levels correlate with longer lifespan [1] . Despite their prevalence, proteome-wide tracking of misincorporations in eukaryotes is challenging, with most insights being recent and coming from the mining of extensive proteomics data sets [2-5] . These experimental challenges have precluded the direct in vivo probing and full understanding of the bases of fidelity in higher eukaryotes, and their influence in complex processes such as aging. Here we measured proteome-wide misincorporation frequencies in two experimentally tractable eukaryotic systems, yeast and mouse, using mass spectrometry. We find translation fidelity is organism and organ specific. The measured error frequencies correlate with codon:anticodon pairing thermodynamics and tRNA pool composition, which can have synergistic or opposing effects on fidelity depending on organism or tissue type. Most identified protein errors carry a negative fitness burden. Aging experiments in our two systems reveal that, with age, error frequencies increase in post-mitotic cells, but not so in mitotic ones. These changes correlate with remodeling of the tRNA pool, which we identify as a crucial regulator of translation fidelity during lifespan.

Aging is characterized by a progressive loss of proteostasis. Transfer RNAs (tRNAs) are essential regulators of translation, yet their dynamics during aging remain poorly understood due to challenges in sequencing highly modified RNAs. Here we present a benchmarked Nanopore direct RNA sequencing (RNA004 chemistry) resource that profiles the Saccharomyces cerevisiae tRNAome during replicative aging at single-molecule resolution. Using in vitro transcribed tRNA controls, we establish modification detection thresholds and validate key findings with orthogonal Illumina sequencing. While overall tRNA abundance remains largely stable, our resource reveals age-associated terminal A cleavage at the 3' CCA tail of mature tRNAs, targeted T-loop and anticodon modification changes, and single-molecule evidence of modification co-occurrence. This dataset provides a resource for exploring tRNA regulation, translation fidelity, and longevity.

N[1]-methylpseudouridine (m[1]Ψ) is a key modification used in SARS-CoV-2 mRNA vaccines that reduces immunogenicity and increases mRNA stability. Recent studies suggested that m[1]Ψ can promote ribosomal frameshifting, a translational error generating aberrant peptides that elicit immune responses, raising concerns about unintended antigenicity. Here, we systematically examined the efficiency of frameshifting induced by m[1]Ψ and the underlying mechanism, aiming to inform future mRNA vaccine design. Using mRNA-based dual-fluorescence reporters in cells, fully in vitro reconstituted translation system, and single-molecule FRET microscopy, we show that m[1]Ψ increases +1 frameshifting on UUUC motifs in therapeutic mRNAs. Frameshifting occurs when the peptidyl-tRNA pauses in the ribosomal P site, where m[1]Ψ both weakens codon-anticodon interactions and promotes a frameshifting-prone tRNA conformation. Replacing slippery UUUC motifs with UUCC or UUUU eliminates this effect. Our results reveal how a clinically relevant mRNA modification promotes recoding and show that codon optimization mitigates this risk in therapeutic mRNA design.

The type VI secretion system (T6SS) is a sophisticated machine that uses a contractile mechanism to deliver antibacterial toxins into neighboring cells, hence participating to interbacterial competition and pathogenesis. Here, we characterize the T6SS-associated rearrangement hotspot RhsF polymorphic toxin from Chromobacterium violaceum. Toxicity assays showed that the RhsF C-terminal extension, TreThr, is a cytoplasmic-acting antibacterial toxin, which is neutralized by the downstream immunity protein. Structural modeling and mutagenesis demonstrated that TreThr belongs to the ADP-ribosyltransferase (ART) family, adopting a canonical ART fold. Using coupled in vitro transcription-translation assays, we found that TreThr inhibits protein synthesis in a Nicotinamide Adenine Dinucleotide (NAD)-dependent manner, through isoacceptor-specific ADP-ribosylation of transfer RNAs (tRNAs). Biotin-NAD labeling experiments and mapping further revealed that TreThr modifies the GGU anticodon of charged thrT and thrV tRNA isoacceptors and prevents decoding of threonine ACC codons.

MTO1 is a nuclear gene that encodes a mitochondrial protein essential for modifying mitochondrial transfer RNAs (tRNAs) and stabilizing codon-anticodon interactions to ensure accurate and efficient mitochondrial protein synthesis and oxidative phosphorylation. Mitochondrial tRNA translation optimization 1 (MTO1) plays an important role in the mitochondrial tRNA taurinomethylation modification by using the amino acid taurine, obtained from cysteine metabolism, at the wobble position U34 of the anticodon loop. Biallelic pathogenic variants in MTO1 cause combined oxidative phosphorylation deficiency 10 (COXPD10) (OMIM#614702). In the severe end of the spectrum, COXPD10 is characterized by infantile-onset hypertrophic cardiomyopathy and lactic acidosis with perinatal mortality when associated with nonsense and frameshift variants. The extra cardiac phenotypes include muscle hypotonia, feeding difficulties, psychomotor delay, optic atrophy, encephalopathy, and seizures. Currently, there is no targeted treatment for this condition aside from supportive care. Herein, we report a 22-month-old child, diagnosed early with a genotype predictive of severe COXPD10, who was initiated on treatment with L-cysteine and N-acetylcysteine (NAC) early in life and did not develop cardiac manifestations. This outcome suggests a potential benefit and improved clinical outcome with early disease-specific treatment initiation.

In eukaryotes, translation initiation is extensively modulated by cis-regulatory sequences located on the mRNA along with trans-acting factors which influence the lifetime of the codon-anticodon interaction in the P site of the scanning 43S complex and eventually control ribosome assembly. Here, we analyzed sequences around AUG start codons to account for mutually dependent or mutually exclusive nucleotide combinations that influence start codon recognition. By comparing the initiation contexts of annotated coding sequences in six model eukaryotes, we show that their nucleotide features are highly species-specific. Searching for accurate consensus nucleotide patterns around AUG start codons identifies multiple nucleotide patterns rather than a single consensus sequence in each eukaryote. Importantly, these patterns highlight distinct nucleotide features around the conserved purine at position -3 across eukaryotes, known to strongly influence initiation efficiency. Finally, computing a species-specific score for any AUG context shows that these diverse nucleotide features associate with specific biological functions.

Aminoacyl-tRNA synthetase complex interacting multifunctional protein 2 (AIMP2) is a component of the eukaryote-specific multisynthetase complex (MSC), a macromolecular assembly of eight aminoacyl-tRNA synthetases (ARS) and three scaffolding components known as aminoacyl-tRNA synthetase interacting multifunctional proteins (AIMP1-3). Recent studies show that bi-allelic mutations in AIMP2 cause a neurodevelopmental syndrome that overlaps phenotypically with disorders caused by recessive ARS mutations. In this work, we further characterize AIMP2 mutations in two patients with severe neurodevelopmental disorders, including one previously unreported compound heterozygous patient. We show patient-derived fibroblast AIMP2 mutations cause decreased protein expression, attenuation of protein synthesis, and dysregulation of the MSC component, methionyl-tRNA synthetase (MARS1). CRISPR-mediated disruption of aimp2 in zebrafish led to increased cell death and atrophy in the developing brain, further supporting a link between AIMP2 dysfunction and neurodevelopmental defects. We tested anticodon engineered suppressor tRNAs on expression of disease-associated nonsense mutations and found that they dramatically increase protein expression of AIMP2 variants. Together, these data demonstrate that AIMP2 depletion leads to neurodevelopmental dysfunction in both human and zebrafish, and suggest that therapies such as anticodon engineered suppressor tRNAs could improve patient outcomes.

In eubacteria, decoding of isoleucine codon AUA requires a specialized tRNA (tRNA[Ile2]) modified with lysidine (k[2]C) at the anticodon wobble position (C34), which switches decoding specificity from methionine (AUG) to isoleucine (AUA). Recently, aminovaleramide cytidine (ava[2]C) was discovered at the same tRNA position in several bacteria and plants and shown to support AUA decoding and Ile-specific aminoacylation. However, the enzyme catalyzing ava[2]C was unknown. Here, we report that tRNA[Ile]-aminovaleramididine synthetase (AvaS) catalyzes ava[2]C biosynthesis in Pseudomonas aeruginosa PA14. AvaS converts k[2]C to ava[2]C through a pyridoxal-phosphate-dependent oxidative decarboxylation mechanism, supported by site-directed mutagenesis and in vitro enzymatic assays. Dual-reporter assays demonstrated that ava[2]C-modified tRNA exhibits lower AUA decoding efficiency than k[2]C-modified tRNA. Additionally, genome-wide screening revealed an unexpected link between ava[2]C levels and metabolic and stress response pathways influencing i[6]A/ms[2]i[6]A dynamics. Together, these findings define the molecular basis of ava[2]C biosynthesis and its broader cellular metabolic networks.

Despite redundant cellular pathways to minimize translational errors, errors in protein synthesis are common. Pathways and mechanisms to minimize errors are classified as pre-ribosomal or ribosomal. Pre-ribosomal pathways are primarily concerned with the appropriate charging of tRNAs with their cognate amino acids. By contrast, the ribosomal decoding center is considered 'blind' to mischarged tRNAs since these have cognate codon•anti-codon pairing. Here, we identified that in mycobacteria, deletion of the 16S ribosomal RNA methyltransferase gidB led to increased ribosomal discrimination of mischarged tRNAs. Discrimination only occurred in mycobacteria enriched from environments or genetic backgrounds with high rates of mistranslation. GidB deletion was necessary, but not sufficient for reducing mistranslation due to misacylation. Analysis of new cryo-EM structures of the M. smegmatis ribosomes derived from wild-type and gidB-deleted strains point to the interaction between the base methylated by GidB on the 16S RNA and an asparagine on the ribosomal S12 protein that, when mistranslated to aspartate, may be involved in altering translational fidelity. Our data suggest a mechanism by which mycobacterial ribosomes can discriminate mischarged tRNAs and that 16S rRNA differential methylation by GidB may act to prevent catastrophic translational error.

Enterococcus faecalis is a gram-positive commensal bacterium of the human gut microbiome and an opportunistic pathogen responsible for many hospital-acquired infections. Despite the clinical importance of E. faecalis, how gene and protein expression are coordinated with growth remains poorly defined. Here, we profiled transcript, protein, and tRNA pool dynamics across distinct phases of E. faecalis growth. Differences in protein abundance and corresponding mRNA levels suggested growth phase-dependent posttranscriptional regulation. Growth-associated genes exhibited biased synonymous codon usage, with ribosomal and glycolytic proteins enriched in low-abundance codons read by queuosine-modifiable tRNAs. Analysis of tRNA modification and tRNA isoacceptor abundance revealed growth phase-dependent changes, particularly in anticodon stem-loop modifications that influence synonymous codon translation. Changes in queuosine levels preceded shifts in ribosomal proteins, suggesting a contribution to codon-biased translation. Collectively, these findings reveal growth phase-associated remodeling of the E. faecalis tRNA pool and support a model in which queuosine-dependent translational reprogramming shapes protein expression during bacterial growth.IMPORTANCEEnterococcus faecalis is a common cause of hospital-acquired infections. Despite its clinical importance, a comprehensive understanding of the organism's physiology and adaptation to environmental changes remains incomplete. Here, we characterized protein, transcript, and tRNA dynamics across bacterial growth phases, uncovering a role for posttranscriptional regulation marked by tRNA reprogramming and biased synonymous codon usage. These findings enhance our understanding of E. faecalis growth and support a model of translational reprogramming therein.

Nucleotide modifications of the tRNA anticodon can affect protein translation fidelity and speed. Chemical modifications of the anticodon nucleotide 34 are regulated under cellular stress and associated with several translational defects and pathologies. Here, we investigate how these modifications influence A-site codon recognition interactions and their coupling to the CAR site that lies adjacent to nucleotide 34 in the ribosome. The conserved three-residue CAR interface hydrogen bonds in a sequence-dependent manner to the mRNA +1 codon 3'-adjacent to the A-site codon and is implicated in tuning translational speed. The C of CAR is pi-stacked with the nucleotide 34 of the A site tRNA anticodon. The A site and the CAR site influence each other's hydrogen bonding and stacking interactions, and these codon-adjacency effects potentially provide a layer of regulation affecting translational fidelity and kinetics. Through molecular dynamics simulations of a subsystem of a translocating ribosome IRES-model, we observed that nucleotide 34 modifications affect the hydrogen bonding and stacking interactions at the A site and CAR site as well as CAR's influence on the A site interactions. Integrating these results with gene sequence and ribosome profiling analyses, we propose that nucleotide 34 modifications help modulate CAR's sequence-dependent tuning of translation in response to cellular stress.

Mutations in mitochondrial tRNA (mt-tRNA) are found to be associated with hypertrophic cardiomyopathy (HCM), but their molecular mechanisms remain largely undetermined. In this study, we investigated the contribution of a novel HCM-related mt-tRNA[Ser(AGY)] 12234A > G mutation to the phenotypic expression of the mt-tRNA[Ile] 4263A > G mutation in two genetically unrelated Han Chinese pedigrees. Strikingly, the penetrance and expressivity of one pedigree (HCM2) with both m.4263A > G and m.12234A > G mutations are much higher than another pedigree (HCM1) with only m.4263A > G mutation. By molecular level, the homoplasmic m.4263A > G mutation is located at the processing site for the tRNA[Ile] 5 [']-end precursor, disrupting a conserved Watson-Crick base pairing (1A-69T) which is believed to cause mitochondrial dysfunction. Moreover, the heteroplasmic m.12234A > G mutation occurs at an extremely conserved nucleotide in the anticodon stem of tRNA[Ser(AGY)], a position which is critical for tRNA structure and function. Using trans-mitochondrial cell models, we demonstrated that cybrids with both mt-tRNA mutations exhibited more severe mitochondrial dysfunctions than cybrids with only the m.4263A > G mutation. Furthermore, a marked decrease in mt-RNA transcripts was observed in cells harboring both m.4263A > G and m.12234A > G mutations. Taken together, our study indicated that the m.12234A > G mutation acted in synergy with the m.4263A > G mutation, triggering mitochondrial dysfunctions and contributing to a high penetrance of HCM in a pedigree harboring both mtDNA mutations.

Post-transcriptional modifications modulate transfer RNA (tRNA) structure, stability, and codon decoding properties, contributing to translation regulation and adaptation across diverse organisms, including bacterial pathogens. We provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry and deep-sequencing methods, generating a high-confidence modification map for each individual tRNA species, including non-proteogenic tRNAGly. While the overall tRNA modification landscape is conserved among Gram-positive bacteria, our data uncovered unexpected S. aureus-specific features. These include the absence of m2A37 in tRNAs despite the presence of the methyltransferase RlmN, a single multi-site DusB2 enzyme catalyzing all tRNA dihydrouridylation, and evidence suggesting a dedicated pseudouridine synthase responsible for Ψ32. Besides, heterogeneous modification patterns were observed in tRNALeu(UAA) and tRNALys(UUU), highlighting a complex interplay in anticodon hypermodification. Time-course proteomics revealed dynamic expression of tRNA modifying enzymes during growth. Integration of ribosome profiling and Nanopore tRNA sequencing offered a global view of S. aureus decoding properties, revealing efficient four-way wobble recognition, slower translation of rare codons by low abundant tRNAs, and distinctive decoding dynamics of Gly codons potentially influenced by the unusual modification status of tRNAGly(UCC). This work establishes a framework to dissect the role of tRNA modifications in S. aureus physiology and pathogenesis.