2005 Articles

Kim HY, Gladyshev VN. (2005) Different Catalytic Mechanisms in Mammalian Selenocysteine- and Cysteine-Containing Methionine-R-Sulfoxide Reductases. PLoS Biol. 3, e375, 1-9.

AbstractSelenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties. More Information

Sun QA, Su D, Novoselov SV, Carlson BA, Hatfield DL, Gladyshev VN. (2005) Reaction mechanism and regulation of Mammalian thioredoxin/glutathione reductase. Biochemistry 44, 14528-14537.

AbstractThioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes. More Information

Castellano S, Lobanov AV, Chapple C, Novoselov SV, Albrecht M, Hua D, Lescure A, Lengauer T, Krol A, Gladyshev VN, Guigo R. (2005) Diversity and functional plasticity of eukaryotic selenoproteins: Identification and characterization of the SelJ family. Proc. Natl. Acad. Sci. 102, 16188-16193.

AbstractSelenoproteins are a diverse group of proteins that contain selenocysteine (Sec), the 21st amino acid. In the genetic code, UGA serves as a termination signal and a Sec codon. This dual role has precluded the automatic annotation of selenoproteins. Recent advances in the computational identification of selenoprotein genes have provided a first glimpse of the size, functions, and phylogenetic diversity of eukaryotic selenoproteomes. Here, we describe the identification of a selenoprotein family named SelJ. In contrast to known selenoproteins, SelJ appears to be restricted to actinopterygian fishes and sea urchin, with Cys homologues only found in cnidarians. SelJ shows significant similarity to the jellyfish J1-crystallins and with them constitutes a distinct subfamily within the large family of ADP-ribosylation enzymes. Consistent with its potential role as a structural crystallin, SelJ has preferential and homogeneous expression in the eye lens in early stages of zebrafish development. A structural role for SelJ would be in contrast to the majority of known selenoenzymes. The unusually highly restricted phylogenetic distribution of SelJ, its specialization, and the comparative analysis of eukaryotic selenoproteomes reveal the diversity and functional plasticity of selenoproteins and point to a mosaic evolution of the use of Sec in proteins. More Information

Xu XM, Mix H, Carlson BA, Grabowski PJ, Gladyshev VN, Berry MJ, Hatfield DL. (2005) Evidence for direct roles of two additional factors, SECp43 and SLA, in the selenoprotein synthesis machinery. J. Biol. Chem. 280, 41568-41575.

AbstractSelenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2′-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA. More Information

Biterova EI, Turanov AA, Gladyshev VN, Barycki JJ. (2005) Crystal structures of oxidized and reduced mitochondrial thioredoxin reductase provide molecular details of the reaction mechanism. Proc. Natl. Acad Sci. 102, 15018-15023.

AbstractThioredoxin reductase (TrxR) is an essential enzyme required for the efficient maintenance of the cellular redox homeostasis, particularly in cancer cells that are sensitive to reactive oxygen species. In mammals, distinct isozymes function in the cytosol and mitochondria. Through an intricate mechanism, these enzymes transfer reducing equivalents from NADPH to bound FAD and subsequently to an active-site disulfide. In mammalian TrxRs, the dithiol then reduces a mobile C-terminal selenocysteine-containing tetrapeptide of the opposing subunit of the dimer. Once activated, the C-terminal redox center reduces a disulfide bond within thioredoxin. In this report, we present the structural data on a mitochondrial TrxR, TrxR2 (also known as TR3 and TxnRd2). Mouse TrxR2, in which the essential selenocysteine residue had been replaced with cysteine, was isolated as a FAD-containing holoenzyme and crystallized (2.6 A; R = 22.2%; R(free) = 27.6%). The addition of NADPH to the TrxR2 crystals resulted in a color change, indicating reduction of the active-site disulfide and formation of a species presumed to be the flavin-thiolate charge transfer complex. Examination of the NADP(H)-bound model (3.0 A; R = 24.1%; R(free) = 31.2%) indicates that an active-site tyrosine residue must rotate from its initial position to stack against the nicotinamide ring of NADPH, which is juxtaposed to the isoalloxazine ring of FAD to facilitate hydride transfer. Detailed analysis of the structural data in conjunction with a model of the unusual C-terminal selenenylsulfide suggests molecular details of the reaction mechanism and highlights evolutionary adaptations among reductases. More Information

Novoselov SV, Calvisi DV, Labunskyy VM, Factor VM, Carlson BA, Fomenko DE, Moustafa ME, Hatfield DL. and Gladyshev VN. (2005) Selenoprotein deficiency and high levels of selenium compounds can effectively inhibit hepatocarcinogenesis in transgenic mice. Oncogene 24, 8003-8011.

AbstractThe micronutrient element selenium (Se) has been shown to be effective in reducing the incidence of cancer in animal models and human clinical trials. Selenoproteins and low molecular weight Se compounds were implicated in the chemopreventive effect, but specific mechanisms are not clear. We examined the role of Se and selenoproteins in liver tumor formation in TGFalpha/c-Myc transgenic mice, which are characterized by disrupted redox homeostasis and develop liver cancer by 6 months of age. In these mice, both Se deficiency and high levels of Se compounds suppressed hepatocarcinogenesis. In addition, both treatments induced expression of detoxification genes, increased apoptosis and inhibited cell proliferation. Within low-to-optimal levels of dietary Se, tumor formation correlated with expression of most selenoproteins. These data suggest that changes in selenoprotein expression may either suppress or promote tumorigenesis depending on cell type and genotype. Since dietary Se may have opposing effects on cancer, it is important to identify the subjects who will benefit from Se supplementation as well as those who will not. More Information

Labunskyy VM, Ferguson AD, Fomenko DE, Chelliah Y, Hatfield DL, Gladyshev VN. (2005) A novel cysteine-rich domain of SEP15 mediates the interactions with UDP-glucose: Glycoprotein glucosyltransferase. J. Biol. Chem. 280, 37839-37845.

AbstractSelenium is an essential trace element with potent cancer prevention activity in mammals. The 15-kDa selenoprotein (Sep15) has been implicated in the chemopreventive effect of dietary selenium. Although the precise function of Sep15 remains elusive, Sep15 co-purifies with UDP-glucose:glycoprotein glucosyltransferase (GT), an essential regulator of quality control mechanisms within the endoplasmic reticulum. Recent studies identified two GT and two Sep15 homologues in mammals. We characterize interactions between these protein families in this report. Sep15 and GT form a tight 1:1 complex, and these interactions are conserved between mammals and fruit flies. In mammalian cells, Sep15 co-immunoprecipitates with both GT isozymes. In contrast, a Sep15 homologue, designated selenoprotein M (SelM), does not form a complex with GT. Sequence analysis of members of the Sep15 family identified a novel N-terminal cysteine-rich domain in Sep15 that is absent in SelM. This domain contains six conserved cysteine residues that form two CxxC motifs that do not coordinate metal ions. If this domain is deleted or the cysteines are mutated, Sep15 no longer forms a complex with GT. Conversely, if the cysteine-rich domain of Sep15 is fused to the N-terminus of SelM, the resulting chimera is capable of binding GT. These data indicate that the cysteine-rich domain of Sep15 exclusively mediates protein-protein interactions with GT. More Information

Romero H, Zhang Y, Gladyshev VN, Salinas G. (2005) Evolution of selenium utilization traits. Genome Biol. 6, R66.

AbstractBACKGROUND: The essential trace element selenium is used in a wide variety of biological processes. Selenocysteine (Sec), the 21st amino acid, is co-translationally incorporated into a restricted set of proteins. It is encoded by an UGA codon with the help of tRNASec (SelC), Sec-specific elongation factor (SelB) and a cis-acting mRNA structure (SECIS element). In addition, Sec synthase (SelA) and selenophosphate synthetase (SelD) are involved in the biosynthesis of Sec on the tRNASec. Selenium is also found in the form of 2-selenouridine, a modified base present in the wobble position of certain tRNAs, whose synthesis is catalyzed by YbbB using selenophosphate as a precursor. RESULTS: We analyzed completely sequenced genomes for occurrence of the selA, B, C, D and ybbB genes. We found that selB and selC are gene signatures for the Sec-decoding trait. However, selD is also present in organisms that do not utilize Sec, and shows association with either selA, B, C and/or ybbB. Thus, selD defines the overall selenium utilization. A global species map of Sec-decoding and 2-selenouridine synthesis traits is provided based on the presence/absence pattern of selenium-utilization genes. The phylogenies of these genes were inferred and compared to organismal phylogenies, which identified horizontal gene transfer (HGT) events involving both traits. CONCLUSION: These results provide evidence for the ancient origin of these traits, their independent maintenance, and a highly dynamic evolutionary process that can be explained as the result of speciation, differential gene loss and HGT. The latter demonstrated that the loss of these traits is not irreversible as previously thought. More Information

Kim HY, Gladyshev VN. (2005) Role of structural and functional elements of mouse methionine-s-sulfoxide reductase in its subcellular distribution. Biochemistry 44, 8059-8067.

AbstractOxidized forms of methionine residues in proteins can be repaired by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). In mammals, three MsrBs are present, which are targeted to various subcellular compartments. In contrast, only a single mammalian MsrA gene is known whose products have been detected in both cytosol and mitochondria. Factors that determine the location of the protein in these compartments are not known. Here, we found that MsrA was present in cytosol, nucleus, and mitochondria in mouse cells and tissues and that the major enzyme forms detected in various compartments were generated from a single-translation product rather than by alternative translation initiation. Both cytosolic and mitochondrial forms were processed with respect to the N-terminal signal peptide, and the distribution of the protein occurred post-translationally. Deletion of amino acids 69-108, 69-83, 84-108, or 217-233, which contained elements important for MsrA structure and function, led to exclusive mitochondrial location of MsrA, whereas a region that affected substrate binding but was not part of the overall fold had no influence on the subcellular distribution. The data suggested that proper structure-function organization of MsrA played a role in subcellular distribution of this protein in mouse cells. These findings were recapitulated by expressing various forms of mouse MsrA in Saccharomyces cerevisiae, suggesting conservation of the mechanisms responsible for distribution of the mammalian enzyme among different cellular compartments. More Information

Su D, Novoselov SV, Sun QA, Moustafa ME, Zhou Y, Oko R, Hatfield DL, Gladyshev VN. (2005) Mammalian selenoprotein thioredoxin/glutathione reductase: Roles in disulfide bond formation and sperm maturation. J. Biol. Chem. 280, 26491-26498.

AbstractThioredoxin reductases (TRs) are important redox regulatory enzymes, which control the redox state of thioredoxins. Mammals have cytosolic and mitochondrial TRs, which contain an essential selenocysteine residue and reduce cytosolic and mitochondrial thioredoxins. In addition, thioredoxin/glutathione reductase (TGR) was identified, which is a fusion of an N-terminal glutaredoxin domain and the TR module. Here we show that TGR is expressed at low levels in various tissues but accumulates in testes after puberty. The protein is particularly abundant in elongating spermatids at the site of mitochondrial sheath formation but is absent in mature sperm. We found that TGR can catalyze isomerization of protein and interprotein disulfide bonds and localized this function to its thiol domain. TGR targets include proteins that form structural components of the sperm, including glutathione peroxidase GPx4/PHGPx. Together, TGR and GPx4 can serve as a novel disulfide bond formation system. Both enzymes contain a catalytic selenocysteine consistent with the role of selenium in male reproduction. More Information

Su D, Li Y, Gladyshev VN. (2005) Selenocysteine insertion directed by the 3′-UTR SECIS element in Escherichia coli. Nucleic Acids Res. 33, 2486-2492.

AbstractCo-translational insertion of selenocysteine (Sec) into proteins in response to UGA codons is directed by selenocysteine insertion sequence (SECIS) elements. In known bacterial selenoprotein genes, SECIS elements are located in the coding regions immediately downstream of UGA codons. Here, we report that a distant SECIS element can also function in Sec insertion in bacteria provided that it is spatially close to the UGA codon. We expressed a mammalian phospholipid hydroperoxide glutathione peroxidase in Escherichia coli from a construct in which a natural E.coli SECIS element was located in the 3′-untranslated region (3′-UTR) and adjacent to a sequence complementary to the region downstream of the Sec UGA codon. Although the major readthrough event at the UGA codon was insertion of tryptophan, Sec was also incorporated and its insertion was dependent on the functional SECIS element in the UTR, base-pairing potential of the SECIS flanking region and the Sec UGA codon. These data provide important implications into evolution of SECIS elements and development of a system for heterologous expression of selenoproteins and show that in addition to the primary sequence arrangement between UGA codons and SECIS elements, their proximity within the tertiary structure can support Sec insertion in bacteria. More Information

Taskov K, Chapple C, Kryukov GV, Castellano S, Lobanov AV, Korotkov KV, Guigó R. and Gladyshev VN. (2005) Nematode selenoproteome: the use of the selenocysteine insertion system to decode one codon in an animal genome? Nucleic Acids Res. 33, 2227-2238.

AbstractSelenocysteine (Sec) is co-translationally inserted into selenoproteins in response to codon UGA with the help of the selenocysteine insertion sequence (SECIS) element. The number of selenoproteins in animals varies, with humans having 25 and mice having 24 selenoproteins. To date, however, only one selenoprotein, thioredoxin reductase, has been detected in Caenorhabditis elegans, and this enzyme contains only one Sec. Here, we characterize the selenoproteomes of C.elegans and Caenorhabditis briggsae with three independent algorithms, one searching for pairs of homologous nematode SECIS elements, another searching for Cys- or Sec-containing homologs of potential nematode selenoprotein genes and the third identifying Sec-containing homologs of annotated nematode proteins. These methods suggest that thioredoxin reductase is the only Sec-containing protein in the C.elegans and C.briggsae genomes. In contrast, we identified additional selenoproteins in other nematodes. Assuming that Sec insertion mechanisms are conserved between nematodes and other eukaryotes, the data suggest that nematode selenoproteomes were reduced during evolution, and that in an extreme reduction case Sec insertion systems probably decode only a single UGA codon in C.elegans and C.briggsae genomes. In addition, all detected genes had a rare form of SECIS element containing a guanosine in place of a conserved adenosine present in most other SECIS structures, suggesting that in organisms with small selenoproteomes SECIS elements may change rapidly. More Information

Zhang Y, Fomenko DE, Gladyshev VN. (2005) The microbial selenoproteome of the Sargasso Sea. Genome Biology 6, R37.

AbstractBACKGROUND: Selenocysteine (Sec) is a rare amino acid which occurs in proteins in major domains of life. It is encoded by TGA, which also serves as the signal for termination of translation, precluding identification of selenoprotein genes by available annotation tools. Information on full sets of selenoproteins (selenoproteomes) is essential for understanding the biology of selenium. Herein, we characterized the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome project. RESULTS: We identified 310 selenoprotein genes that clustered into 25 families, including 101 new selenoprotein genes that belonged to 15 families. Most of these proteins were predicted redox proteins containing catalytic selenocysteines. Several bacterial selenoproteins previously thought to be restricted to eukaryotes were detected by analyzing eukaryotic and bacterial SECIS elements, suggesting that eukaryotic and bacterial selenoprotein sets partially overlapped. The Sargasso Sea microbial selenoproteome was rich in selenoproteins and its composition was different from that observed in the combined set of completely sequenced genomes, suggesting that these genomes do not accurately represent the microbial selenoproteome. Most detected selenoproteins occurred sporadically compared to the widespread presence of their cysteine homologs, suggesting that many selenoproteins recently evolved from cysteine-containing homologs. CONCLUSIONS: This study yielded the largest selenoprotein dataset to date, doubled the number of prokaryotic selenoprotein families and provided insights into forces that drive selenocysteine evolution. More Information 

See commentary in:Copeland PR. (2005) Making sense of nonsense: the evolution of selenocysteine usage in proteins. Genome Biol., 6, 221. Commentary
Zhang Y, Gladyshev VN. (2005) An algorithm for identification of bacterial selenocysteine insertion sequence elements and selenoprotein genes. Bioinformatics 21, 2580-2589.

AbstractMOTIVATION: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec insertion sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking. RESULTS: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in sequence databases. The program identified 96.5% of known selenoprotein genes in completely sequenced bacterial genomes and predicted several new selenoprotein genes. Further analysis revealed that the size of bacterial selenoproteomes varied from 1 to 11 selenoproteins. Formate dehydrogenase was present in most selenoproteomes, often as the only selenoprotein family, whereas the occurrence of other selenoproteins was limited. The availability of the bacterial SECIS consensus and the tool for identification of these structures should help in correct annotation of selenoprotein genes and characterization of bacterial selenoproteomes. More Information

Zhang Y, Baranov PV, Atkins JF, Gladyshev VN. (2005) Pyrrolysine and selenocysteine use dissimilar decoding strategies. J. Biol. Chem. 280, 20740-20751.

AbstractSelenocysteine (Sec) and pyrrolysine (Pyl) are known as the 21st and 22nd amino acids in protein. Both are encoded by codons that normally function as stop signals. Sec specification by UGA codons requires the presence of a cis-acting selenocysteine insertion sequence (SECIS) element. Similarly, it is thought that Pyl is inserted by UAG codons with the help of a putative pyrrolysine insertion sequence (PYLIS) element. Herein, we analyzed the occurrence of Pyl-utilizing organisms, Pyl-associated genes, and Pyl-containing proteins. The Pyl trait is restricted to several microbes, and only one organism has both Pyl and Sec. We found that methanogenic archaea that utilize Pyl have few genes that contain in-frame UAG codons, and many of these are followed with nearby UAA or UGA codons. In addition, unambiguous UAG stop signals could not be identified. This bias was not observed in Sec-utilizing organisms and non-Pyl-utilizing archaea, as well as with other stop codons. These observations as well as analyses of the coding potential of UAG codons, overlapping genes, and release factor sequences suggest that UAG is not a typical stop signal in Pyl-utilizing archaea. On the other hand, searches for conserved Pyl-containing proteins revealed only four protein families, including methylamine methyltransferases and transposases. Only methylamine methyltransferases matched the Pyl trait and had conserved Pyl, suggesting that this amino acid is used primarily by these enzymes. These findings are best explained by a model wherein UAG codons may have ambiguous meaning and Pyl insertion can effectively compete with translation termination for UAG codons obviating the need for a specific PYLIS structure. Thus, Sec and Pyl follow dissimilar decoding and evolutionary strategies. More Information

Shrimali RK, Lobanov AV, Xu XM, Rao M, Carlson BA, Mahadeo DC, Parent CA, Gladyshev VN, Hatfield DL. (2005) Selenocysteine tRNA identification in the model organisms Dictyostelium discoideum and Tetrahymena thermophila. Biochem. Biophys. Res. Commun. 329, 147-151.

AbstractCharacterizing Sec tRNAs that decode UGA provides one of the most direct and easiest means of determining whether an organism possesses the ability to insert selenocysteine (Sec) into protein. Herein, we used a combination of two techniques, computational to identify Sec tRNA genes and RT-PCR to sequence the gene products, to unequivocally demonstrate that two widely studied, model protozoans, Dictyostelium discoideum and Tetrahymena thermophila, encode Sec tRNA in their genomes. The advantage of using both procedures is that computationally we could easily detect potential Sec tRNA genes and then confirm by sequencing that the Sec tRNA was present in the tRNA population, and thus the identified gene was not a pseudogene. Sec tRNAs from both organisms decode UGA. T. thermophila Sec tRNA, like all other sequenced Sec tRNAs, is 90 nucleotides in length, while that from D. discoideum is 91 nucleotides long making it the longest eukaryotic sequenced to date. Evolutionary analyses of known Sec tRNAs reveal the two forms identified herein are the most divergent eukaryotic Sec tRNAs thus far sequenced. More Information

Carlson BA, Xu XM, Gladyshev VN, Hatfield DL. (2005) Selective rescue of selenoprotein expression in mice lacking a highly specialized methyl group in selenocysteine tRNA. J. Biol. Chem. 280, 5542-5548.

AbstractSelenocysteine (Sec) is the 21st amino acid in the genetic code. Its tRNA is variably methylated on the 2′-O-hydroxyl site of the ribosyl moiety at position 34 (Um34). Herein, we identified a role of Um34 in regulating the expression of some, but not all, selenoproteins. A strain of knock-out transgenic mice was generated, wherein the Sec tRNA gene was replaced with either wild type or mutant Sec tRNA transgenes. The mutant transgene yielded a tRNA that lacked two base modifications, N(6)-isopentenyladenosine at position 37 (i(6)A37) and Um34. Several selenoproteins, including glutathione peroxidases 1 and 3, SelR, and SelT, were not detected in mice rescued with the mutant transgene, whereas other selenoproteins, including thioredoxin reductases 1 and 3 and glutathione peroxidase 4, were expressed in normal or reduced levels. Northern blot analysis suggested that other selenoproteins (e.g. SelW) were also poorly expressed. This novel regulation of protein expression occurred at the level of translation and manifested a tissue-specific pattern. The available data suggest that the Um34 modification has greater influence than the i(6)A37 modification in regulating the expression of various mammalian selenoproteins and Um34 is required for synthesis of several members of this protein class. Many proteins that were poorly rescued appear to be involved in responses to stress, and their expression is also highly dependent on selenium in the diet. Furthermore, their mRNA levels are regulated by selenium and are subject to nonsense-mediated decay. Overall, this study described a novel mechanism of regulation of protein expression by tRNA modification that is in turn regulated by levels of the trace element, selenium. More Information