Base 37 in tRNA, 3′-adjacent to the anticodon, is occupied by a purine base that is thought to stabilize codon recognition by stacking interactions on the first Watson-Crick base pair. If the first codon position forms an A.U or U.A base pair, the purine is likely further modified in all domains of life. One of the first base modifications found in tRNA is N⁶-isopentenyl adenosine (i⁶A) present in a fraction of tRNAs in bacteria and eukaryotes, which can be further modified to 2-methyl-thio-N⁶-isopentenyladenosine (ms²i⁶A) in a subset of tRNAs. Homologous tRNA isopentenyl transferase enzymes have been identified in bacteria (MiaA), yeast (Mod5, Tit1), roundworm (GRO-1), and mammals (TRIT1). In eukaryotes, isopentenylation of cytoplasmic and mitochondrial tRNAs is mediated by products of the same gene. Accordingly, a patient with homozygous mutations in TRIT1 has mitochondrial disease. The role of i⁶A in a subset of tRNAs in gene expression has been linked with translational fidelity, speed of translation, skewed gene expression, and non-sense suppression. This review will not cover the action of i⁶A as a cytokinin in plants or the potential function of Mod5 as a prion in yeast.

Transfer RNA^[Ser]Sec carries multiple post-transcriptional modifications. The A37G mutation in tRNA^[Ser]Sec abrogates isopentenylation of base 37 and has a profound effect on selenoprotein expression in mice. Patients with a homozygous pathogenic p.R323Q variant in tRNA-isopentenyl-transferase (TRIT1) show a severe neurological disorder, and hence we wondered whether selenoprotein expression was impaired. Patient fibroblasts with the homozygous p.R323Q variant did not show a general decrease in selenoprotein expression. However, recombinant human TRIT1^R323Q had significantly diminished activities towards several tRNA substrates in vitro. We thus engineered mice conditionally deficient in Trit1 in hepatocytes and neurons. Mass-spectrometry revealed that hypermodification of U₃₄ to mcm⁵Um occurs independently of isopentenylation of A₃₇ in tRNA^[Ser]Sec. Western blotting and ⁷⁵Se metabolic labeling showed only moderate effects on selenoprotein levels and ⁷⁵Se incorporation. A detailed analysis of Trit1-deficient liver using ribosomal profiling demonstrated that UGA/Sec re-coding was moderately affected in Selenop, Txnrd1, and Sephs2, but not in Gpx1. 2′O-methylation of U₃₄ in tRNA^[Ser]Sec depends on FTSJ1, but does not affect UGA/Sec re-coding in selenoprotein translation. Taken together, our results show that a lack of isopentenylation of tRNA^[Ser]Sec affects UGA/Sec read-through but differs from a A37G mutation.

Selenoproteins are a small family of proteins containing the trace element selenium in form of the rare amino acid selenocysteine (Sec), which is decoded by the UGA codon. In humans, a number of pathogenic variants in genes encoding distinct selenoproteins or selenoprotein biosynthesis factors have been identified. Pathogenic variants in selenocysteine synthase (SEPSECS), which catalyzes the last step in Sec-tRNA^[Ser]Sec biosynthesis, were reported in children suffering from progressive cerebello-cerebral atrophy. To understand the pathomechanism associated with SEPSECS deficiency, we generated a novel mouse model recapitulating the respective human pathogenic p.Y334C variant in the murine Sepsecs gene (Sepsecs^Y334C). Unlike in patients, pups homozygous for the p.Y334C variant died perinatally with signs of cardio-respiratory failure. Perinatal death is reminiscent of the Sedaghatian spondylometaphyseal dysplasia disorder in humans, which is caused by pathogenic variants in the gene encoding the selenoprotein and key ferroptosis regulator glutathione peroxidase 4 (GPX4). Protein expression levels of distinct selenoproteins in Sepsecs^Y334C/Y334C mice were found to be generally reduced in brain and isolated cortical neurons, while transcriptomics analysis uncovered an upregulation of NRF2-regulated genes. Crossbreeding of Sepsecs^Y334C/Y334C mice with mice harboring a targeted mutation of the catalytically active Sec to Cys in GPX4 rescued perinatal death of Sepsecs^Y334C/Y334C mice, showing that the cardio-respiratory defects of Sepsecs^Y334C/Y334C mice were caused by the lack of GPX4. Like in Sepsecs^Y334C/Y334C mice, selenoprotein expression levels remained low and NRF2-regulated genes remained highly expressed in these compound mutant mice, indicating that selenium-independent GPX4, along with a sustained antioxidant response are sufficient to compensate for dysfunctional Sec-tRNA^[Ser]Sec biosynthesis. Our findings imply that children with pathogenic variants in SEPSECS or GPX4 may even benefit from treatments that incompletely compensate for impaired GPX4 activity.

Co-translational incorporation of selenocysteine (Sec) into selenoproteins occurs at UGA codons in a process in which translational elongation competes with translational termination. Selenocysteine insertion sequence-binding protein 2 (SECISBP2) greatly enhances Sec incorporation into selenoproteins by interacting with the mRNA, ribosome, and elongation factor Sec (EFSEC). Ribosomal profiling allows to study the process of UGA re-coding in the physiological context of the cell and at the same time for all individual selenoproteins expressed in that cell. Using HAP1 cells expressing a mutant SECISBP2, we show here that high-resolution ribosomal profiling can be used to assess read-through efficiency at the UGA in all selenoproteins, including those with Sec close to the C-terminus. Analysis of ribosomes with UGA either at the A-site or the P-site revealed, in a transcript-specific manner, that SECISBP2 helps to recruit tRNA^Sec and stabilize the mRNA. We propose to assess the effect of any perturbation of UGA read-through by determining the proportion of ribosomes carrying UGA in the P-site, pUGA. An additional, new observation is frameshifting that occurred 3′ of the UGA/Sec codon in SELENOF and SELENOW in SECISBP2-mutant HAP1 cells, a finding corroborated by reanalysis of neuron-specific Secisbp2^R543Q-mutant brains.

Vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) catalyzes the reduction of vitamin K within the endoplasmic reticulum (ER) and has recently been shown to suppress ferroptosis. However, its physiological role remains largely unknown. Here, we show that Vkorc1l1-/- mice spontaneously developed progressive stages of steatotic liver disease (SLD) on a chow diet, independent of metabolic dysfunction. Mechanistically, despite the loss of VKORC1L1-mediated ferroptosis suppression, Vkorc1l1-/- mice activated compensatory NRF2-driven antioxidant responses and enhanced hepatic triacylglycerol (TAG) synthesis. These adaptations promoted the sequestration of polyunsaturated fatty acids into lipid droplets (LDs), thereby limiting ferroptosis-induced hepatic injury. Furthermore, metabolism of Vkorc1l1-/- mice was reprogrammed to a chronic fasting-like phenotype characterized by with enhanced mitochondrial function. Notably, the radical-trapping antioxidant menaquinone-4, but not phylloquinone, both of which are substrates of VKORC1L1, decreased lipid droplet number, whereas inhibition of VKORC1L1 through warfarin enhanced LD biogenesis in HepG2 cells. In humans, a common VKORC1L1 risk haplotype was associated with reduced hepatic VKORC1L1 expression and with increased risk of metabolic dysfunction-associated steatotic liver disease in the UK Biobank and alcohol-associated liver cirrhosis in case–control cohorts. This work identifies VKORC1L1 as a key regulator of SLD pathogenesis by orchestrating metabolic rewiring to maintain redox homeostasis and suppress ferroptosis in vivo.