Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA[Ser]Sec and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA [Ser]Sec , termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA[Ser]Sec -EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.Significant strides have been made over the past 15 years in elucidating the mechanism and most of the players in eukaryotic selenoprotein biosynthesis. Key players in this process are the unique tRNA that decodes UGA as a selenocysteine codon (16), the specific secondary structures in the 3Ј untranslated regions of selenoprotein mRNAs, termed SECIS elements, that are required for selenocysteine insertion (2), and protein factors that interact with the tRNA and SECIS element. Protein factors identified to date include an elongation factor specific for selenocysteyl (Sec)-tRNA [Ser]Sec , termed EFsec (10, 26), the SECIS binding protein, SBP2 (6), and most recently, a ribosomal protein, L30, that can also bind SECIS elements and may mediate the incorporation process at the ribosome (5). Two selenophosphate synthetases, SPS1 and SPS2, contribute to the selenoprotein synthesis pathway, in that they catalyze conversion of selenide and ATP to selenophosphate, the active selenium donor in selenocysteine biosynthesis (18). SPS2 is itself a selenoenzyme, thus serving a positive feedback role in selenoprotein synthesis. Recently, a kinase that phosphorylates Ser-tRNA [Ser]Sec has been identified in the genomes of organisms that encode other components of the selenoprotein synthesis machinery (4). However, its role in this process remains to be elucidated.At least two activities crucial to selenocysteine incorporation have remained elusive, the factors(s) responsible for conversion of Ser-tRNA [Ser]Sec to Sec-tRNA . Tw...

The Y chromosome is thought to be important for male reproduction. We have previously shown that with the use of assisted reproduction, live offspring can be obtained from mice lacking the entire Y chromosome long arm. Here, we demonstrated that live mouse progeny can also be generated using germ cells from males with the Y chromosome contribution limited to only two genes, the testis determinant factor Sry and the spermatogonial proliferation factor Eif2s3y. Sry is believed to function primarily in sex determination during fetal life. Eif2s3y may be the only Y chromosome gene required to drive mouse spermatogenesis allowing formation of haploid germ cells that are functional in assisted reproduction. Our findings are relevant but not directly translatable to human male infertility cases.

Selenoprotein H is a recently identified member of the selenoprotein family whose function is not fully known. Previous studies from our laboratory and others showed that Drosophila melanogaster selenoprotein H is essential for viability and antioxidant defense. In this study we investigated the function of human selenoprotein H in murine hippocampal HT22 cells engineered to stably overexpress the protein. After treatment of cells with L-buthionine-(S,R)-sulfoximine to deplete glutathione, selenoprotein H-overexpressing cells exhibited higher levels of total glutathione, total antioxidant capacities, and glutathione peroxidase enzymatic activity than did vector control cells. Overexpression of selenoprotein H also up-regulated the mRNA levels of endogenous selenoprotein H, glutamylcysteine synthetase heavy and light chains, and glutathione S-transferases Alpha 2, Alpha 4, and Omega 1. The amino acid sequence of selenoprotein H contains four putative nuclear localization sequences and an AT-hook motif, a small DNA-binding domain first identified in high mobility group proteins. Chromatin immunoprecipitation using a green fluorescent protein-selenoprotein H fusion revealed binding to sequences containing heat shock and/or stress response elements. Thus, selenoprotein H is a redox-responsive DNA-binding protein of the AT-hook family and functions in regulating expression levels of genes involved in de novo glutathione synthesis and phase II detoxification in response to redox status.Selenium has long been known for its antioxidant properties, and accumulated evidence indicates that many of the beneficial effects of this trace element in our diet are attributable to selenoenzymes. The functions of selenoenzymes include protecting cell membranes, proteins, and nucleic acids from cumulative oxidative damage and maintaining cellular redox balance. Selenium is highly retained in neuronal tissue during selenium deficiency (1), and the functions of selenoproteins in the brain are highlighted by the development of neurological defects in mice that underwent targeted disruption of selenoprotein P, a selenium transport protein whose functions may also include antioxidant defense and heavy metal chelation (2, 3). To date, 25 selenoprotein genes have been identified in the human genome (4), but the functions of many of them are yet to be fully defined. Selenoprotein H (SelH) 3 was initially identified in the Drosophila melanogaster genome and subsequently in the human and mouse genomes, where expression is high in the brain. In previous studies we and others showed that D. melanogaster SelH is required for viability, and overexpression of the protein increased antioxidant capacity in Drosophila embryo-derived Schneider S2 cells when exposed to homocysteic acid-induced GSH depletion (5, 6).Two recent studies have begun to investigate the functions of human SelH. We reported that overexpression of human SelH protects against UV-induced cell death via a decrease in superoxide levels (7). A study employing bioinformatic analysis a...

Selenocysteine is incorporated into proteins via "recoding" of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3 untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a "bottleneck," slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.Selenoprotein P is an enigma of genetic recoding. It is the only known protein in which multiple potential stop codons are recoded to function as sense codons. In selenoprotein mRNAs, UGA codons, which would normally signal the termination of protein synthesis, are decoded as the amino acid selenocysteine. This process involves a unique tRNA with an anticodon complementary to UGA and specialized secondary structures located in the 3Ј untranslated regions (UTRs) of selenoprotein mRNAs, termed selenocysteine incorporation sequence (SECIS) elements (3). For selenocysteine incorporation to occur, the SECIS element must recruit a SECIS binding protein, SBP2 (6). SBP2, in turn, recruits a dedicated elongation factor, EFsec (7, 26), complexed with selenocysteyl-tRNA (2, 31). Most selenoprotein mRNAs contain a single UGA codon and a single SECIS element. Selenoprotein P mRNAs contain 10 to 18 UGA codons, depending on the species, and two SECIS elements differing in secondary structure. The majority of studies to date on the mechanism of selenoprotein synthesis have focused on selenoprotein mRNAs containing a single UGA codon and a single SECIS element. Thus, little is known about the mechanism for incorporating multiple selenocysteines.The incorporation of selenium into selenoprotein P serves several important biological functions: it allows the accumulation of this essential but potentially toxic trace element in a biologically stable and nontoxic form, it plays a critical role in the transport of selenium from the liver via the circulation to target organs (13,25), it is thought to function in sequestering and thereby detoxifying heavy metals in plasma...