tRNA Sec Research Articles

A homozygous mutation in the human selenocysteine tRNA gene impairs UGA recoding activity and selenoproteome regulation by selenium.

The selenocysteine (Sec) tRNA (tRNA[Ser]Sec) governs Sec insertion into selenoproteins by the recoding of a UGA codon, typically used as a stop codon. A homozygous point mutation (C65G) in the human tRNA[Ser]Sec acceptor arm has been reported by two independent groups and was associated with symptoms such as thyroid dysfunction and low blood selenium levels; however, the extent of altered selenoprotein synthesis resulting from this mutation has yet to be comprehensively investigated. In this study, we used CRISPR/Cas9 technology to engineer homozygous and heterozygous mutant human cells, which we then compared with the parental cell lines. This C65G mutation affected many aspects of tRNA[Ser]Sec integrity and activity. Firstly, the expression level of tRNA[Ser]Sec was significantly reduced due to an altered recruitment of RNA polymerase III at the promoter. Secondly, selenoprotein expression was strongly altered, but, more surprisingly, it was no longer sensitive to selenium supplementation. Mass spectrometry analyses revealed a tRNA isoform with unmodified wobble nucleotide U34 in mutant cells that correlated with reduced UGA recoding activities. Overall, this study demonstrates the pleiotropic effect of a single C65G mutation on both tRNA phenotype and selenoproteome expression.

Cul5-type Ubiquitin Ligase KLHDC1 Contributes to the Elimination of Truncated SELENOS Produced by Failed UGA/Sec Decoding.

SummaryThe UGA codon signals protein translation termination, but it can also be translated into selenocysteine (Sec, U) to produce selenocysteine-containing proteins (selenoproteins) by dedicated machinery. As Sec incorporation can fail, Sec-containing longer and Sec-lacking shorter proteins co-exist. Cul2-type ubiquitin ligases were recently shown to destabilize such truncated proteins; however, which ubiquitin ligase targets truncated proteins for degradation remained unclear. We report that the Cul5-type ubiquitin ligase KLHDC1 targets truncated SELENOS, a selenoprotein, for proteasomal degradation. SELENOS is involved in endoplasmic reticulum (ER)-associated degradation, which is linked to reactive oxygen species (ROS) production, and the knockdown of KLHDC1 in U2OS cells decreased ER stress-induced cell death. Knockdown of SELENOS increased the cell population with lower ROS levels. Our findings reveal that, in addition to Cul2-type ubiquitin ligases, KLHDC1 is involved in the elimination of truncated oxidoreductase-inactive SELENOS, which would be crucial for maintaining ROS levels and preventing cancer development.

Pelodiscus sinensis embryos infected with Aeromonas hydrophila show higher survival rates than infected Mauremys reevesiis embryos

Emydidae and Trionychidae used different strategies to adapt to the environmental condition. However, the differences in the immune responses are not yet fully understood. In this study, Pelodiscus sinensis (P. sinensis) embryos infected with Aeromonas hydrophila (A. hydrophila) showed significantly higher survival rates than infected Mauremys reevesiis (M. reevesiis) embryos. Transcriptomic profiles of infected and non-infected M. reevesiis and P. sinensis embryos were explored. In total, 8,358 differentially expressed unigenes were detected, of which 3,294 and 5,064 genes were considered to be up- and down-regulated in P. sinensis compared to those in M. reevesiis. Six differentially expressed genes (DEGs) were randomly selected for further validation by qRT-PCR, and the results agreed with the RNA-seq data. Analysing the transcriptome data, eight significantly up/down-regulated immune genes (basic transcription factor 3 (BTF3); patched domain containing 1 (PTCHD1); Sep (O-phosphoserine) tRNA: Sec (selenocysteine) tRNA synthase (SEPSECS); zinc finger FYVE-type containing 16 (ZFYVE16); EF-hand domain family member D2 (EFHD2); zinc finger MYM-type containing 4 (ZMYM4); histone deacetylase 9 (HDAC9); interleukin 10 receptor subunit beta (IL-10RB)) were found. Expression patterns of eight selected genes after A. hydrophila injection were detected via qRT-PCR. In the early developmental stages of infected embryos, HDAC9 and IL-10RB significantly up-regulated which play a key role in the activation of NK cells and Th cells in P. sinensis, while M. reevesiis up-regulated BTF3 and ZFYVE16 first to start proliferation of B-lymphoid cells. We provide new insights into the immunity difference between Emydidae and Trionychidae, facilitating the development of new methods to treat bacterial infections in turtles.

Designing seryl-tRNA synthetase for improved serylation of selenocysteine tRNAs.

Selenocysteine (Sec) lacks a cognate aminoacyl-tRNA synthetase. Instead, seryl-tRNA synthetase (SerRS) produces Ser-tRNASec , which is subsequently converted by selenocysteine synthase to Sec-tRNASec . Escherichia coli SerRS serylates tRNASec poorly; this may hinder efficient production of designer selenoproteins invivo. Guided by structural modelling and selection for chloramphenicol acetyltransferase activity, we evolved three SerRS variants capable of improved Ser-tRNASec synthesis. They display 10-, 8-, and 4-fold increased kcat /KM values compared to wild-type SerRS using synthetic tRNASec species as substrates. The enzyme variants also facilitate invivo read-through of a UAG codon in the position of the critical serine146 of chloramphenicol acetyltransferase. These results indicate that the naturally evolved SerRS is capable of further evolution for increased recognition of a specific tRNA isoacceptor.

Selenoproteins regulate stress erythroid progenitors and spleen microenvironment during stress erythropoiesis

AbstractMicronutrient selenium (Se) plays a key role in redox regulation through its incorporation into selenoproteins as the 21st amino acid selenocysteine (Sec). Because Se deficiency appears to be a cofactor in the anemia associated with chronic inflammatory diseases, we reasoned that selenoproteins may contribute to erythropoietic recovery from anemia, referred to as stress erythropoiesis. Here, we report that loss of selenoproteins through Se deficiency or by mutation of the Sec tRNA (tRNA[Sec]) gene (Trsp) severely impairs stress erythropoiesis at 2 stages. Early stress erythroid progenitors failed to expand and properly differentiate into burst-forming unit-erythroid cells, whereas late-stage erythroid progenitors exhibited a maturation defect that affected the transition of proerythroblasts to basophilic erythroblasts. These defects were, in part, a result of the loss of selenoprotein W (SelenoW), whose expression was reduced at both transcript and protein levels in Se-deficient erythroblasts. Mutation of SelenoW in the bone marrow cells significantly decreased the expansion of stress burst-forming unit-erythroid cell colonies, which recapitulated the phenotypes induced by Se deficiency or mutation of Trsp. Similarly, mutation of SelenoW in murine erythroblast (G1E) cell line led to defects in terminal differentiation. In addition to the erythroid defects, the spleens of Se-deficient mice contained fewer red pulp macrophages and exhibited impaired development of erythroblastic island macrophages, which make up the niche supporting erythroblast development. Taken together, these data reveal a critical role of selenoproteins in the expansion and development of stress erythroid progenitors, as well as the erythroid niche during acute anemia recovery.

Selenocysteine tRNA[Ser]Sec, the Central Component of Selenoprotein Biosynthesis: Isolation, Identification, Modification, and Sequencing.

The selenocysteine (Sec) tRNA[Ser]Sec population consists of two isoforms that differ from each other by a single 2'-O-methylribosyl moiety at position 34 (Um34). These two isoforms, which are encoded in a single gene, Trsp, and modified posttranscriptionally, are involved individually in the synthesis of two subclasses of selenoproteins, designated housekeeping and stress-related selenoproteins. Techniques used in obtaining these isoforms for their characterization include extraction of RNA from mammalian cells and tissues, purifying the tRNA[Ser]Sec population by one or more procedures, and finally resolving the two isoforms from each other. Since some of the older techniques for isolating tRNA[Ser]Sec and resolving the isoforms are used in only a few laboratories, these procedures will be discussed briefly and references provided for more detailed information, while the more recently developed procedures are discussed in detail. In addition, a novel technique that was developed in sequencing tRNA[Ser]Sec for identifying their occurrence in other organisms is also presented.

Dynamics and Energetics of Elongation Factor SelB in the Ternary Complex and the Ribosome

SelB is an elongation factor specialized to deliver the selenocysteine (Sec) tRNA to the ribosome by recoding the UGA stop codon on the mRNA. Initially the tRNA is in complex with selB and GTP forming the ternary complex (TC). High-resolution cryo-EM structures of intermediates of the Sec incorporation pathway uncover large-scale conformational changes of the ribosome and the TC. To complement the structural information with energetics and rapid dynamics, we performed extensive all-atom molecular dynamics simulations of the ribosome with bound TC as well as of the free TC in solution. The simulations of the free TC were started after extracting the TC from the ribosome-bound cryo-EM structures. The TC was found to rapidly interconvert between the different conformations allowing us to construct the free-energy landscape of the involved motions. This free-energy landscape indicates that the intrinsic large-scale conformational changes of the tRNA and SelB during the delivery to the ribosome are not rate-limiting to the process. In simulations of the free TC started from the GTPase-activated ribosome-bound conformation, the TC rapidly transitions into an inactivated conformation, showing that the GTPase-activated state is strongly stabilized by the ribosome. The simulations of the full ribosome with bound TC in the intermediate states allow us to identify the motions that are rate-limiting to the process of tRNA delivery and to identify the molecular mechanism of the domain closure of small ribosomal subunit upon tRNA decoding.

Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration

The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4−/− mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4−/− livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4−/− pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4−/− mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.

Removal of Glutathione Peroxidase 4 in Mouse Liver Leads to Early Lethality and Vitamin E Can Compensate for Its Loss

Glutathione peroxidase 4 (GPx4) is one of several glutathione peroxidases found in mammals and its targeted deletion is early embryonic lethal. The primary functions of GPx4 are to protect cells against membrane lipid peroxidation and to govern a novel form of non‐apoptotic cell death, called ferroptosis, which appears to be relevant in a select set of organs. To study the role of GPx4 in liver tissue, a conditional knockout of Gpx4 in hepatocytes was used, along with thioredoxin reductase 1 (TrxR1) and selenocysteine (Sec) tRNA (Trsp) hepatocyte knockout mice as controls. In contrast to TrxR1‐ and Trsp‐deficient mice, Gpx4 knockout mice died within 48 hours after birth and presented extensive hepatocyte degeneration. We further found that Gpx4 knockout pups born from mothers fed a vitamin E‐enriched diet did not experience early death, demonstrating that vitamin E rescued Gpx4 knockout‐induced cell death. When vitamin E was subsequently removed from the diet, Gpx4‐deficient mice had an average survival time of approximately four weeks. We also crossed Trsp‐deficient and Gpx4‐deficient mice and were unable to obtain combined Trsp/Gpx4‐deficient mice at birth, indicating that the combined deficiency resulted in embryonic death. Combined Trsp/TrxR1‐deficient mice were born but had significantly shorter lifespans than either single knockout, suggesting that TrxR1 plays an important role in longevity of mice lacking Trsp in hepatocytes. This study demonstrates that GPx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against lipid peroxidation.Support or Funding InformationThis work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Milder progressive cerebellar atrophy caused by biallelic SEPSECS mutations.

Cerebellar atrophy is recognized in various types of childhood neurological disorders with clinical and genetic heterogeneity. Genetic analyses such as whole exome sequencing are useful for elucidating the genetic basis of these conditions. Pathological recessive mutations in Sep (O-phosphoserine) tRNA:Sec (selenocysteine) tRNA synthase (SEPSECS) have been reported in a total of 11 patients with pontocerebellar hypoplasia type 2, progressive cerebellocerebral atrophy or progressive encephalopathy, yet detailed clinical features are limited to only four patients. We identified two new families with progressive cerebellar atrophy, and by whole exome sequencing detected biallelic SEPSECS mutations: c.356A>G (p.Asn119Ser) and c.77delG (p.Arg26Profs*42) in family 1, and c.356A>G (p.Asn119Ser) and c.467G>A (p.Arg156Gln) in family 2. Their development was slightly delayed regardless of normal brain magnetic resonance imaging (MRI) in infancy. The progression of clinical symptoms in these families is evidently slower than in previously reported cases, and the cerebellar atrophy milder by brain MRI, indicating that SEPSECS mutations are also involved in milder late-onset cerebellar atrophy.

Identification of Determinants that Govern Processive and Efficient Selenocysteine (Sec) Incorporation

Selenoproteins include essential proteins and are unique as they incorporate selenocysteine (Sec), the 21st amino acid, in response to an stop codon (UGA). Thus selenoprotein expression is a challenge as it requires stop codon redefinition. To date, five essential factors for Sec incorporation have been identified. This includes the mRNA with an inframe UGA codon, a stem loop structure referred to as SECIS element located in the 3′UTR, a dedicated elongation factor (eEFSec), a SECIS binding protein (SBP2) and a unique Sec tRNA Sec‐tRNASec. However the mechanistic details of Sec incorporation still remains unclear. Among the known selenoproteins, Selenoprotein P (SEPP1) is unique mainly as it contains multiple UGA codons and possesses two SECIS elements in the 3′UTR. Thus, full length SEPP1 synthesis requires processive Sec incorporation which in vivo appears to be efficient. The focus of this research is to identify core determinants that govern processive and efficient Sec incorporation. Using 75Se labeling in vitro we demonstrate that selenium supplementation stimulates processive Sec incorporation by 4‐fold thus linking active Sec‐tRNASec synthesis with processive Sec incorporation. We also establish that while the SEPP1 mRNA is inherently processive for Sec incorporation, there exists an interplay between the coding region and the 3′UTR that governs the degree of processivity. Furthermore cellular transfection studies indicate RNA stability as a crucial determinant of efficiency in mammalian SEPP1 synthesis. Thus translation regulation primarily governs processive and efficient Sec incorporation via channeling of Sec incorporation components and also by recruiting novel cis elements.

The selenocysteine tRNA gene in leishmania major is transcribed by both RNA polymerase II and RNA polymerase III.

Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major. This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5' end of all the mRNAs. In L. major, tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5' rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5' end and a poly(A) tail at the 3' end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei. However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo, as has recently been found in other eukaryotes.

Investigation of Escherichia coli Selenocysteine Synthase (SelA) Complex Formation Using Cryo-Electron Microscopy (Cryo-EM)

Incorporation of selenocysteine (Sec U) into proteins is directed by a in-frame UGA codon in all domains of life. In Bacteria, Sec biosynthesis and incorporation involves the interaction of Selenocysteine Synthase (SelA), tRNA (SelC or tRNA Sec ), Selenophosphate Synthetase (SPS), a specific elongation factor known as SelB and the specific mRNA structure SElenocysteine Insertion Sequence (SECIS), forming a complex molecular machinery. SelA is a homodecamer complex responsible for Ser-Sec conversion from selenophosphate delivered by SPS and seryl-tRNA sec , which differs from seryl-tRNA ser by its long variable arm and the UGA-codon. The specific mRNA sequence known as SElenoCysteine Insertion Sequence forms a hairpin-like secondary structure and is recognized by SelB for Sec incorporation in the nascent peptide [1-3]. Since selenium compounds are highly toxic in cellular environment, selenium association with proteins complexes throughout its metabolism is suggested to be essential for cell survival. However, macromolecular interactions between the different proteins have not yet been characterized.

Prostate Epithelium-Specific Deletion of the Selenocysteine tRNA Gene Trsp Leads to Early Onset Intraepithelial Neoplasia

Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.

Tertiary structure of bacterial selenocysteine tRNA

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNASec) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNASec from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNASec assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNASecs, A. aeolicus tRNASec has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNASec in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNASec. Similar interactions exist in the reported tRNASer and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNASec in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNASec to enter the SerRS catalytic site.

High Error Rates in Selenocysteine Insertion in Mammalian Cells Treated with the Antibiotic Doxycycline, Chloramphenicol, or Geneticin

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.

Interaction of mineral salts with the skin: a literature survey

There is growing scientific evidence that the health, well-being and the attractiveness of the skin are strongly influenced by nutrition. Consumers recognize this and have supported the creation of a global cosmeceuticals market estimated in 2010 at $27.2 billion. Early in 2011, the US Department of Health and Human Services and Department of Agriculture issued the Dietary Guidelines for Americans, 2010. Twelve vitamins and nine minerals were recognized as essential. The minerals include calcium, copper, iron, magnesium, phosphorus, selenium, zinc, potassium and sodium. Although the topical benefits of several minerals such as zinc, magnesium and iron are recognized and, in some cases, approved by the FDA, the topical benefits of the others to the skin are largely unexplored and unexploited. This review attempts to summarize what has been published in the literature on the interactions of the eight of the nine essential elements with the skin.

Crystallization and preliminary X-ray crystallographic analysis of bacterial tRNA(Sec) in complex with seryl-tRNA synthetase.

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA(Sec)) is first ligated with serine by seryl-tRNA synthetase (SerRS). To elucidate the tertiary structure of bacterial tRNA(Sec) and its specific interaction with SerRS, the bacterial tRNA(Sec) from Aquifex aeolicus was crystallized as the heterologous complex with the archaeal SerRS from Methanopyrus kandleri. Although X-ray diffraction by crystals of tRNA(Sec) in complex with wild-type SerRS was rather poor (to 5.7 Å resolution), the resolution was improved by introducing point mutations targeting the crystal-packing interface. Heavy-atom labelling also contributed to resolution improvement. A 3.2 Å resolution diffraction data set for phase determination was obtained from a K(2)Pt(CN)(4)-soaked crystal.

Thyroid Function Is Maintained Despite Increased Oxidative Stress in Mice Lacking Selenoprotein Biosynthesis in Thyroid Epithelial Cells

We have tested the hypothesis that selenium (Se)-containing antioxidative enzymes protect thyroid epithelial cells from oxidative damage associated with enzymatic production of hydrogen peroxide required for thyroid hormone biosynthesis. Thyroid epithelial cells therefore express antioxidative enzymes, including catalase, peroxiredoxins, thioredoxin reductases, and glutathione peroxidases (GPxs). The latter two enzyme families contain highly active peroxide-degrading enzymes that carry selenocysteine (Sec) in their active centers. Since low Se status has been associated with thyroid disorders, selenoproteins are considered essential for thyroid integrity and function. We have conditionally inactivated selenoprotein biosynthesis in thyrocytes by targeting Sec tRNA. Constitutive and inducible Cre/loxP-mediated recombination of tRNA([Ser]Sec) drastically reduced activities of selenoenzymes GPx and type I-deiodinase in thyroid extracts. Immunohistochemical staining revealed increased 4-hydroxynonenal and 3-nitro-tyrosine levels consistent with increased oxidative stress. However, gross thyroid morphology remained intact for at least 6 months after recombination. Circulating thyroid hormone levels remained normal in mutant mice, while thyrotropin (TSH) levels were moderately elevated. Challenging mutant mice with low iodine diet increased TSH, but did not lead to destruction of selenoprotein-deficient thyroids. This is the first report probing the assumed physiological roles of selenoproteins in the thyroid using a genetic loss-of-function approach. We conclude that selenoproteins protect thyrocytes from oxidative damage and modulate thyroid hormone biosynthesis, but are not essential for thyrocyte survival.

Mammalian selenocysteine tRNA Um34 methylase and its role in selenoprotein synthesis

Selenocysteine (Sec) tRNA plays a key role in recoding the UGA stop codon to insert the 21st amino acid, Sec, into selenoproteins (SPs). The mammalian Sec tRNA population consists of two isoforms that differ by a single methyl group, 2’‐O‐methylribose, at the nucleotide 34 wobble position in the anticodon (designated Um34). Um34 synthesis is a highly specialized event in mammalian Sec tRNA maturation and is involved in the synthesis of a subgroup of SPs implicated in stress‐related phenomena. We used a comparative genomic approach and knockdown/knock‐in techniques to uncover a purported Um34 methylase. Knockdown of Um34 methylase in NIH3T3 cells resulted in decreased glutathione peroxidase 1 (GPx1, a stress‐related SP) expression. Over‐expression of this methylase led to an increase in GPx1 synthesis. Um34 methylase synthesis is regulated by selenium status. The data suggest that Sec tRNA Um34 methylation is carried out by the purported methylase. Further investigation of this methylase may provide new avenues to modulate SP synthesis, and further assess its role in disease including certain cancers. Funding: Intramural Research Program, NIH, NCI, CCR and DFG grants to U.S. and L.S.