Proteolytic cleavage and inactivation of the TRMT1 tRNA modification enzyme by SARS-CoV-2 main protease.

Nonstructural protein 5 (Nsp5) is the main protease of SARS-CoV-2 that cleaves viral polyproteins into individual polypeptides necessary for viral replication. Here, we show that Nsp5 binds and cleaves human tRNA methyltransferase 1 (TRMT1), a host enzyme required for a prevalent post-transcriptional modification in tRNAs. Human cells infected with SARS-CoV-2 exhibit a decrease in TRMT1 protein levels and TRMT1-catalyzed tRNA modifications, consistent with TRMT1 cleavage and inactivation by Nsp5. Nsp5 cleaves TRMT1 at a specific position that matches the consensus sequence of SARS-CoV-2 polyprotein cleavage sites, and a single mutation within the sequence inhibits Nsp5-dependent proteolysis of TRMT1. The TRMT1 cleavage fragments exhibit altered RNA binding activity and are unable to rescue tRNA modification in TRMT1-deficient human cells. Compared to wildtype human cells, TRMT1-deficient human cells infected with SARS-CoV-2 exhibit reduced levels of intracellular viral RNA. These findings provide evidence that Nsp5-dependent cleavage of TRMT1 and perturbation of tRNA modification patterns contribute to the cellular pathogenesis of SARS-CoV-2 infection.

tRNA modification enzyme-dependent redox homeostasis regulates synapse formation and memory.

Post-transcriptional modification of RNA regulates gene expression at multiple levels. ALKBH8 is a tRNA modifying enzyme that methylates wobble uridines in specific tRNAs to modulate translation. Through methylation of tRNA-selenocysteine, ALKBH8 promotes selenoprotein synthesis and regulates redox homeostasis. Pathogenic variants in ALKBH8 have been linked to intellectual disability disorders in the human population, but the role of ALKBH8 in the nervous system is unknown. Through in vivo studies in Drosophila, we show that ALKBH8 controls oxidative stress in the brain to restrain synaptic growth and support learning and memory. ALKBH8 null animals lack wobble uridine methylation and exhibit a global reduction in protein synthesis, including a specific decrease in selenoprotein levels. Loss of ALKBH8 or independent disruption of selenoprotein synthesis results in ectopic synapse formation. Genetic expression of antioxidant enzymes fully suppresses synaptic overgrowth in ALKBH8 null animals, confirming oxidative stress as the underlying cause of dysregulation. ALKBH8 animals also exhibit associative learning and memory impairments that are reversed by pharmacological antioxidant treatment. Together, these findings demonstrate the critical role of tRNA modification in redox homeostasis in the nervous system and reveal antioxidants as a potential therapy for ALKBH8-associated intellectual disability.

Expanded tRNA methyltransferase family member TRMT9B regulates synaptic growth and function.

Nervous system function rests on the formation of functional synapses between neurons. We have identified TRMT9B as a new regulator of synapse formation and function in Drosophila. TRMT9B has been studied for its role as a tumor suppressor and is one of two metazoan homologs of yeast tRNA methyltransferase 9 (Trm9), which methylates tRNA wobble uridines. Whereas Trm9 homolog ALKBH8 is ubiquitously expressed, TRMT9B is enriched in the nervous system. However, in the absence of animal models, TRMT9B's role in the nervous system has remained unstudied. Here, we generate null alleles of TRMT9B and find it acts postsynaptically to regulate synaptogenesis and promote neurotransmission. Through liquid chromatography-mass spectrometry, we find that ALKBH8 catalyzes canonical tRNA wobble uridine methylation, raising the question of whether TRMT9B is a methyltransferase. Structural modeling studies suggest TRMT9B retains methyltransferase function and, in vivo, disruption of key methyltransferase residues blocks TRMT9B's ability to rescue synaptic overgrowth, but not neurotransmitter release. These findings reveal distinct roles for TRMT9B in the nervous system and highlight the significance of tRNA methyltransferase family diversification in metazoans.

ADATscan - A flexible tool for scanning exomes for wobble inosine-dependent codons reveals a neurological bias for genes enriched in such codons in humans and mice.

The conversion of adenosine to inosine at the wobble position of select tRNAs is essential for decoding specific codons in bacteria and eukarya. In eukarya, wobble inosine modification is catalyzed by the heterodimeric ADAT complex containing ADAT2 and ADAT3. Human individuals homozygous for loss of function variants in ADAT3 exhibit intellectual disability disorders. We created a flexible computational tool to scan the human, mouse, nematode, fruit fly, and yeast exomes for genes either enriched or depleted in ADAT-dependent codons as compared to background models of codon bias derived from the exomes themselves. We find that many genes are enriched or depleted for ADAT-dependent codons as compared to the genomic background in all five species. Among those genes enriched for ADAT-dependent codons in humans, we find there is significant Gene Ontology (GO) enrichment for genes involved in diverse neurological processes. This pattern persists in the mouse exome but not the fruit fly or nematode exome. In the nematode exome, genes enriched in ADAT-dependent codons are GO enriched for translation associated genes, and in yeast there is GO enrichment for genes involved in metabolic functions. There is also GO-term overlap between yeast and fruit flies. Importantly, in its generalized form, ADATscan can also be used to scan any exome for genes enriched in any subset of codons specified by the user.

Methyltransferase METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs.

A subset of eukaryotic tRNAs is methylated in the anticodon loop, forming 3-methylcytosine (m3C) modifications. In mammals, the number of tRNAs containing m3C modifications has been expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. However, whereas the enzymes catalyzing m3C formation in nuclear-encoded tRNAs have been identified, the proteins responsible for m3C modification in mt-tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase, as well as with mt-tRNAs containing m3C. We demonstrate that human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs, but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells could be rescued by re-expression of WT METTL8, but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, we found METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA, suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mt-tRNAs and uncover a potential function for m3C modification in mitochondrial tRNA structure.

THUMPD1 bi-allelic variants cause loss of tRNA acetylation and a syndromic neurodevelopmental disorder.

Covalent tRNA modifications play multi-faceted roles in tRNA stability, folding, and recognition, as well as the rate and fidelity of translation, and other cellular processes such as growth, development, and stress responses. Mutations in genes that are known to regulate tRNA modifications lead to a wide array of phenotypes and diseases including numerous cognitive and neurodevelopmental disorders, highlighting the critical role of tRNA modification in human disease. One such gene, THUMPD1, is involved in regulating tRNA N4-acetylcytidine modification (ac4C), and recently was proposed as a candidate gene for autosomal-recessive intellectual disability. Here, we present 13 individuals from 8 families who harbor rare loss-of-function variants in THUMPD1. Common phenotypic findings included global developmental delay, speech delay, moderate to severe intellectual deficiency, behavioral abnormalities such as angry outbursts, facial dysmorphism, and ophthalmological abnormalities. We demonstrate that the bi-allelic variants identified cause loss of function of THUMPD1 and that this defect results in a loss of ac4C modification in small RNAs, and of individually purified tRNA-Ser-CGA. We further corroborate this effect by showing a loss of tRNA acetylation in two CRISPR-Cas9-generated THUMPD1 KO cell lines. In addition, we also show the resultant amino acid substitution that occurs in a missense THUMPD1 allele identified in an individual with compound heterozygous variants results in a marked decrease in THUMPD1 stability and RNA-binding capacity. Taken together, these results suggest that the lack of tRNA acetylation due to THUMPD1 loss of function results in a syndromic form of intellectual disability associated with developmental delay, behavioral abnormalities, hearing loss, and facial dysmorphism.

Detection of tRNA-specific adenosine deaminase activity and wobble inosine modification in human cell lysates.

The wobble inosine modification plays a central role in translation by enabling a single tRNA to decode multiple synonymous codons. In eukaryotes, the formation of wobble inosine is catalyzed by a heterodimeric adenosine deaminase complex comprised of the ADAT2 and ADAT3 subunits. Notably, pathogenic variants in the ADAT3 subunit have been identified as the cause of autosomal recessive intellectual disability in the human population by impacting wobble inosine levels. Here, we describe approaches for monitoring adenosine deaminase activity and inosine modification status at the wobble position of cellular tRNAs. To detect adenosine deaminase activity, we provide protocols for preparing extracts from human cells followed by enzymatic assays with in vitro transcribed tRNA substrates. Furthermore, we describe a method to monitor wobble inosine status of individual tRNAs using cDNA sequencing. These assays can be used to decipher the molecular basis for neurodevelopmental disorders linked to wobble inosine deficiency and disease-associated ADAT2/3 variants.

Monitoring the 5-Methoxycarbonylmethyl-2-Thiouridine (mcm5s2U) Modification Utilizing the Gamma-Toxin Endonuclease.

The post-transcriptional modification of tRNAs at the wobble position plays a critical role in proper mRNA decoding and efficient protein synthesis. In particular, certain wobble uridines in eukaryotes are converted to 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The mcm5s2U modification modulates decoding during translation by increasing the stringency of the wobble uridine to base pair with its canonical nucleotide partner, thereby restricting decoding to its cognate codon. Here, we outline a technique to monitor wobble uridine status in mcm5s2U-containing tRNAs using the gamma-toxin endonuclease from the yeast Kluyveromyces lactis that naturally cleaves tRNAs containing the mcm5s2U modification. This technique is coupled to Northern blotting or reverse transcription-PCR to enable rapid and sensitive detection of changes in mcm5s2U modification state.

ALKBH7 mediates necrosis via rewiring of glyoxal metabolism.

Alkb homolog 7 (ALKBH7) is a mitochondrial α-ketoglutarate dioxygenase required for DNA alkylation-induced necrosis, but its function and substrates remain unclear. Herein, we show ALKBH7 regulates dialdehyde metabolism, which impacts the cardiac response to ischemia-reperfusion (IR) injury. Using a multi-omics approach, we find no evidence ALKBH7 functions as a prolyl-hydroxylase, but we do find Alkbh7-/- mice have elevated glyoxalase I (GLO-1), a dialdehyde detoxifying enzyme. Metabolic pathways related to the glycolytic by-product methylglyoxal (MGO) are rewired in Alkbh7-/- mice, along with elevated levels of MGO protein adducts. Despite greater glycative stress, hearts from Alkbh7-/- mice are protected against IR injury, in a manner blocked by GLO-1 inhibition. Integrating these observations, we propose ALKBH7 regulates glyoxal metabolism, and that protection against necrosis and cardiac IR injury bought on by ALKBH7 deficiency originates from the signaling response to elevated MGO stress.

Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder.

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the ADAT3 locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived from two ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA.

DALRD3 encodes a protein mutated in epileptic encephalopathy that targets arginine tRNAs for 3-methylcytosine modification.

In mammals, a subset of arginine tRNA isoacceptors are methylated in the anticodon loop by the METTL2 methyltransferase to form the 3-methylcytosine (m3C) modification. However, the mechanism by which METTL2 identifies specific tRNA arginine species for m3C formation as well as the biological role of m3C in mammals is unknown. Here, we show that human METTL2 forms a complex with DALR anticodon binding domain containing 3 (DALRD3) protein to recognize particular arginine tRNAs destined for m3C modification. DALRD3-deficient human cells exhibit nearly complete loss of the m3C modification in tRNA-Arg species. Notably, we identify a homozygous nonsense mutation in the DALRD3 gene that impairs m3C formation in human patients exhibiting developmental delay and early-onset epileptic encephalopathy. These findings uncover an unexpected function for the DALRD3 protein in the targeting of distinct arginine tRNAs for m3C modification and suggest a crucial biological role for DALRD3-dependent tRNA modification in proper neurological development.

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.

An intellectual disability-associated missense variant in TRMT1 impairs tRNA modification and reconstitution of enzymatic activity.

The human TRMT1 gene encodes an RNA methyltransferase enzyme responsible for catalyzing dimethylguanosine (m2,2G) formation in transfer RNAs (tRNAs). Frameshift mutations in TRMT1 have been shown to cause autosomal-recessive intellectual disability (ID) in the human population but additional TRMT1 variants remain to be characterized. Here, we describe a homozygous TRMT1 missense variant in a patient displaying developmental delay, ID, and epilepsy. The missense variant changes an arginine residue to a cysteine (R323C) within the methyltransferase domain and is expected to perturb protein folding. Patient cells expressing TRMT1-R323C exhibit a deficiency in m2,2G modifications within tRNAs, indicating that the mutation causes loss of function. Notably, the TRMT1 R323C mutant retains tRNA binding but is unable to rescue m2,2G formation in TRMT1-deficient human cells. Our results identify a pathogenic point mutation in TRMT1 that perturbs tRNA modification activity and demonstrate that m2,2G modifications are disrupted in the cells of patients with TRMT1-associated ID disorders.

Biallelic variants in CTU2 cause DREAM-PL syndrome and impair thiolation of tRNA wobble U34.

The wobble position in the anticodon loop of transfer ribonucleic acid (tRNA) is subject to numerous posttranscriptional modifications. In particular, thiolation of the wobble uridine has been shown to play an important role in codon-anticodon interactions. This modification is catalyzed by a highly conserved CTU1/CTU2 complex, disruption of which has been shown to cause abnormal phenotypes in yeast, worms, and plants. We have previously suggested that a single founder splicing variant in human CTU2 causes a novel multiple congenital anomalies syndrome consisting of dysmorphic facies, renal agenesis, ambiguous genitalia, microcephaly, polydactyly, and lissencephaly (DREAM-PL). In this study, we describe five new patients with DREAM-PL phenotype and whose molecular analysis expands the allelic heterogeneity of the syndrome to five different alleles; four of which predict protein truncation. Functional characterization using patient-derived cells for each of these alleles, as well as the original founder allele; revealed a specific impairment of wobble uridine thiolation in all known thiol-containing tRNAs. Our data establish a recognizable CTU2-linked autosomal recessive syndrome in humans characterized by defective thiolation of the wobble uridine. The potential deleterious consequences for the translational efficiency and fidelity during development as a mechanism for pathogenicity represent an attractive target of future investigations.

Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability.

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine-to-methionine mutation (V144M) in ADAT3 that originated ∼1,600 years ago is the most common cause of autosomal recessive intellectual disability (ID) in Arabia. While the mutation is predicted to affect protein structure, the molecular and cellular effects of the V144M mutation are unknown. Here, we show that cell lines derived from ID-affected individuals expressing only ADAT3-V144M exhibit decreased wobble inosine in certain tRNAs. Moreover, extracts from the same cell lines of ID-affected individuals display a severe reduction in tRNA deaminase activity. While ADAT3-V144M maintains interactions with ADAT2, the purified ADAT2/3-V144M complexes exhibit defects in activity. Notably, ADAT3-V144M exhibits an increased propensity to form aggregates associated with cytoplasmic chaperonins that can be suppressed by ADAT2 overexpression. These results identify a key role for ADAT2-dependent folding of ADAT3 in wobble inosine modification and indicate that proper formation of an active ADAT2/3 complex is crucial for proper neurodevelopment.

The emerging impact of tRNA modifications in the brain and nervous system.

A remarkable number of neurodevelopmental disorders have been linked to defects in tRNA modifications. These discoveries place tRNA modifications in the spotlight as critical modulators of gene expression pathways that are required for proper organismal growth and development. Here, we discuss the emerging molecular and cellular functions of the diverse tRNA modifications linked to cognitive and neurological disorders. In particular, we describe how the structure and location of a tRNA modification influences tRNA folding, stability, and function. We then highlight how modifications in tRNA can impact multiple aspects of protein translation that are instrumental for maintaining proper cellular proteostasis. Importantly, we describe how perturbations in tRNA modification lead to a spectrum of deleterious biological outcomes that can disturb neurodevelopment and neurological function. Finally, we summarize the biological themes shared by the different tRNA modifications linked to cognitive disorders and offer insight into the future questions that remain to decipher the role of tRNA modifications. This article is part of a Special Issue entitled: mRNA modifications in gene expression control edited by Dr. Soller Matthias and Dr. Fray Rupert.

Phosphorylation of human TRM9L integrates multiple stress-signaling pathways for tumor growth suppression.

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.

Monitoring the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification in eukaryotic tRNAs via the γ-toxin endonuclease.

The post-transcriptional modification of tRNA at the wobble position is a universal process occurring in all domains of life. In eukaryotes, the wobble uridine of particular tRNAs is transformed to the 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) modification which is critical for proper mRNA decoding and protein translation. However, current methods to detect mcm5s2U are technically challenging and/or require specialized instrumental expertise. Here, we show that γ-toxin endonuclease from the yeast Kluyveromyces lactis can be used as a probe for assaying mcm5s2U status in the tRNA of diverse eukaryotic organisms ranging from protozoans to mammalian cells. The assay couples the mcm5s2U-dependent cleavage of tRNA by γ-toxin with standard molecular biology techniques such as northern blot analysis or quantitative PCR to monitor mcm5s2U levels in multiple tRNA isoacceptors. The results gained from the γ-toxin assay reveals the evolutionary conservation of the mcm5s2U modification across eukaryotic species. Moreover, we have used the γ-toxin assay to verify uncharacterized eukaryotic Trm9 and Trm112 homologs that catalyze the formation of mcm5s2U. These findings demonstrate the use of γ-toxin as a detection method to monitor mcm5s2U status in diverse eukaryotic cell types for cellular, genetic, and biochemical studies.

Elp3 and RlmN: A tale of two mitochondrial tail-anchored radical SAM enzymes in Toxoplasma gondii.

Radical S-adenosylmethionine (rSAM) enzymes use a 5'-deoxyadensyl 5'-radical to methylate a wide array of diverse substrates including proteins, lipids and nucleic acids. One such enzyme, Elongator protein-3 (TgElp3), is an essential protein in Toxoplasma gondii, a protozoan parasite that can cause life-threatening opportunistic disease. Unlike Elp3 homologues which are present in all domains of life, TgElp3 localizes to the outer mitochondrial membrane (OMM) via a tail-anchored trafficking mechanism in Toxoplasma. Intriguingly, we identified a second tail-anchored rSAM domain containing protein (TgRlmN) that also localizes to the OMM. The transmembrane domain (TMD) on Toxoplasma Elp3 and RlmN homologues is required for OMM localization and has not been seen beyond the chromalveolates. Both TgElp3 and TgRlmN contain the canonical rSAM amino acid sequence motif (CxxxCxxC) necessary to form the 4Fe-4S cluster required for tRNA modifications. In E. coli, RlmN is responsible for the 2-methlyadenosine (m2A) synthesis at purine 37 in tRNA while in S. cerevisiae, Elp3 is necessary for the formation of 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) at the wobble tRNA position. To investigate why these two rSAM enzymes localize to the mitochondrion in Toxoplasma, and whether or not TgRlmN and TgElp3 possess tRNA methyltransferase activity, a series of mutational and biochemical studies were performed. Overexpression of either TgElp3 or TgRlmN resulted in a significant parasite replication defect, but overexpression was tolerated if either the TMD or rSAM domain was mutated. Furthermore, we show the first evidence that Toxoplasma tRNAGlu contains the mcm5s2U modification, which is the putative downstream product generated by TgElp3 activity.

TRMT1-Catalyzed tRNA Modifications Are Required for Redox Homeostasis To Ensure Proper Cellular Proliferation and Oxidative Stress Survival.

Mutations in the tRNA methyltransferase 1 (TRMT1) gene have been identified as the cause of certain forms of autosomal-recessive intellectual disability (ID). However, the molecular pathology underlying ID-associated TRMT1 mutations is unknown, since the biological role of the encoded TRMT1 protein remains to be determined. Here, we have elucidated the molecular targets and function of TRMT1 to uncover the cellular effects of ID-causing TRMT1 mutations. Using human cells that have been rendered deficient in TRMT1, we show that TRMT1 is responsible for catalyzing the dimethylguanosine (m2,2G) base modification in both nucleus- and mitochondrion-encoded tRNAs. TRMT1-deficient cells exhibit decreased proliferation rates, alterations in global protein synthesis, and perturbations in redox homeostasis, including increased endogenous ROS levels and hypersensitivity to oxidizing agents. Notably, ID-causing TRMT1 variants are unable to catalyze the formation of m2,2G due to defects in RNA binding and cannot rescue oxidative stress sensitivity. Our results uncover a biological role for TRMT1-catalyzed tRNA modification in redox metabolism and show that individuals with TRMT1-associated ID are likely to have major perturbations in cellular homeostasis due to the lack of m2,2G modifications.