The Effect of tRNA[Ser]Sec Isopentenylation on Selenoprotein Expression.

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 TRIT1R323Q 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.

Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations.

Recoding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3'-UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNA-binding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA recoding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2.

High-Resolution Ribosome Profiling Reveals Gene-Specific Details of UGA Re-Coding in Selenoprotein Biosynthesis.

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 tRNASec 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 Secisbp2R543Q-mutant brains.

Missense mutation in selenocysteine synthase causes cardio-respiratory failure and perinatal death in mice which can be compensated by selenium-independent GPX4.

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 (SepsecsY334C). 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 SepsecsY334C/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 SepsecsY334C/Y334C mice with mice harboring a targeted mutation of the catalytically active Sec to Cys in GPX4 rescued perinatal death of SepsecsY334C/Y334C mice, showing that the cardio-respiratory defects of SepsecsY334C/Y334C mice were caused by the lack of GPX4. Like in SepsecsY334C/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.

Modeling the Biochemical Phenotype of MCT8 Mutations In Vitro: Resolving a Troubling Inconsistency.

Allan-Herndon-Dudley syndrome (AHDS) is a severe genetic disease caused by mutations in the monocarboxylate transporter 8 (MCT8) gene. MCT8 mediates transport of thyroid hormones in and out of cells, which is thought to play a pivotal role for embryonic and postnatal development of the human brain. Disconcertingly, MCT8R271H leads to a severe form of AHDS but shows residual transport activity when expressed in several types of cultured cells. Here we try to determine the mechanism behind the transport function of MCT8R271H found in overexpressing cell systems. Mutations of Arg271 were introduced into human MCT8 and stably transfected into Madin-Darby canine kidney cells and the human-derived cell line JEG1. Radioactive thyroid hormone-uptake experiments were performed to analyze the pH-dependent effect of the mutation on transport activity. Arg271His transports thyroid hormones in and out of cells in a pH-dependent manner. Its transport activity increases below pH 7.3 and is clearly diminished at physiological pH. The Michaelis constant of the mutant is unaltered, whereas the maximum velocity is reduced. The expression of Arg271His in JEG1 cells leads to an almost nonfunctional transporter at physiological pH replicating the human phenotype for this mutant in vitro and demonstrates, again, that mutant MCT8 activity depends on cellular background. The protonation of His271 at acidic pH restores activity of the mutant protein, which is not active in its deprotonated form at physiological pH. Thus, experimental parameters must be controlled carefully when modeling MCT8 deficiency in cells.

A dominant vimentin mutant upregulates Hsp70 and the activity of the ubiquitin-proteasome system, and causes posterior cataracts in transgenic mice.

Vimentin is the main intermediate filament (IF) protein of mesenchymal cells and tissues. Unlike other IF-/- mice, vimentin-/- mice provided no evidence of an involvement of vimentin in the development of a specific disease. Therefore, we generated two transgenic mouse lines, one with a (R113C) point mutation in the IF-consensus motif in coil1A and one with the complete deletion of coil 2B of the rod domain. In epidermal keratins and desmin, point mutations in these parts of the alpha-helical rod domain cause keratinopathies and desminopathies, respectively. Here, we demonstrate that substoichiometric amounts of vimentin carrying the R113C point mutation disrupted the endogenous vimentin network in all tissues examined but caused a disease phenotype only in the eye lens, leading to a posterior cataract that was paralleled by the formation of extensive protein aggregates in lens fibre cells. Unexpectedly, central, postmitotic fibres became depleted of aggregates, indicating that they were actively removed. In line with an increase in misfolded proteins, the amounts of Hsp70 and ubiquitylated vimentin were increased, and proteasome activity was raised. We demonstrate here for the first time that the expression of mutated vimentin induces a protein-stress response that contributes to disease pathology in mice, and hypothesise that vimentin mutations cause cataracts in humans.