Selenocysteine incorporation directed from the 3'UTR: characterization of eukaryotic EFsec and mechanistic implications.

The mechanism of selenocysteine incorporation in eukaryotes has been assumed for almost a decade to be inherently different from that in prokaryotes, due to differences in the architecture of selenoprotein mRNAs in the two kingdoms. After extensive efforts in a number of laboratories spanning the same time frame, some of the essential differences between these mechanisms are finally being revealed, through identification of the factors catalyzing cotranslational selenocysteine insertion in eukaryotes. A single factor in prokaryotes recognizes both the selenoprotein mRNA, via sequences in the coding region, and the unique selenocysteyl-tRNA, via both its secondary structure and amino acid.

Selenium metabolism in Drosophila: selenoproteins, selenoprotein mRNA expression, fertility, and mortality.

Selenocysteine is a rare amino acid in protein that is encoded by UGA with the requirement of a downstream mRNA stem-loop structure, the selenocysteine insertion sequence element. To detect selenoproteins in Drosophila, the entire genome was analyzed with a novel program that searches for selenocysteine insertion sequence elements, followed by selenoprotein gene signature analyses. This computational screen and subsequent metabolic labeling with (75)Se and characterization of selenoprotein mRNA expression resulted in identification of three selenoproteins: selenophosphate synthetase 2 and novel G-rich and BthD selenoproteins that had no homology to known proteins.

Nasal meningioma: report of one case and review.

A case of primary nasal meningioma in a 69-year-old women is described. The pathologic, radiologic and clinical characteristics are described. A summary of previously published articles on the subject is given.

Selective inhibition of selenocysteine tRNA maturation and selenoprotein synthesis in transgenic mice expressing isopentenyladenosine-deficient selenocysteine tRNA.

Selenocysteine (Sec) tRNA (tRNA([Ser]Sec)) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA([Ser]Sec) lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA([Ser]Sec) genes into the mouse genome. Overexpression of wild-type tRNA([Ser]Sec) did not affect selenoprotein synthesis.

Distribution and functional consequences of nucleotide polymorphisms in the 3'-untranslated region of the human Sep15 gene.

Selenium has been shown to prevent cancer in a variety of animal model systems. Both epidemiological studies and supplementation trials have supported its efficacy in humans. However, the mechanism by which selenium suppresses tumor development remains unknown.

Association between the 15-kDa selenoprotein and UDP-glucose:glycoprotein glucosyltransferase in the endoplasmic reticulum of mammalian cells.

Mammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding.

Decoding apparatus for eukaryotic selenocysteine insertion.

Decoding UGA as selenocysteine requires a unique tRNA, a specialized elongation factor, and specific secondary structures in the mRNA, termed SECIS elements. Eukaryotic SECIS elements are found in the 3' untranslated region of selenoprotein mRNAs while those in prokaryotes occur immediately downstream of UGA. Consequently, a single eukaryotic SECIS element can serve multiple UGA codons, whereas prokaryotic SECIS elements only function for the adjacent UGA, suggesting distinct mechanisms for recoding in the two kingdoms.

1-Methylguanosine in place of Y base at position 37 in phenylalanine tRNA is responsible for its shiftiness in retroviral ribosomal frameshifting.

Many mammalian retroviruses express their protease and polymerase by ribosomal frameshifting. It was originally proposed that a specialized shifty tRNA promotes the frameshift event. We previously observed that phenylalanine tRNA(Phe) lacking the highly modified wybutoxosine (Y) base on the 3' side of its anticodon stimulated frameshifting, demonstrating that this tRNA is shifty.

Heterogeneity within animal thioredoxin reductases. Evidence for alternative first exon splicing.

Animal thioredoxin reductases (TRs) are selenocysteine-containing flavoenzymes that utilize NADPH for reduction of thioredoxins and other protein and nonprotein substrates. Three types of mammalian TRs are known, with TR1 being a cytosolic enzyme, and TR3, a mitochondrial enzyme. Previously characterized TR1 and TR3 occurred as homodimers of 55-57-kDa subunits.

Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA[Ser]Sec is governed by both primary and tertiary structure.

The selenocysteine (Sec) tRNA[Ser]Sec population in higher vertebrates consists of two major isoacceptors that differ from each other by a single nucleoside modification in the wobble position of the anticodon (position 34). One isoacceptor contains 5-methylcarboxymethyluridine (mcmU) in this position, whereas the other contains 5-methylcarboxymethyluridine-2'-O-methylribose (mcmUm). The other modifications in these tRNAs are N6-isopentenyladenosine (i6A), pseudouridine (psi), and 1-methyladenosine (m1A) at positions 37, 55, and 58, respectively.

Structure-expression relationships of the 15-kDa selenoprotein gene. Possible role of the protein in cancer etiology.

Selenium has been implicated in cancer prevention, but the mechanism and possible involvement of selenoproteins in this process are not understood. To elucidate whether the 15-kDa selenoprotein may play a role in cancer etiology, the complete sequence of the human 15-kDa protein gene was determined, and various characteristics associated with expression of the protein were examined in normal and malignant cells and tissues. The 51-kilobase pair gene for the 15-kDa selenoprotein consisted of five exons and four introns and was localized on chromosome 1p31, a genetic locus commonly mutated or deleted in human cancers.

Multiple levels of regulation of selenoprotein biosynthesis revealed from the analysis of human glioma cell lines.

To gain a better understanding of the biological consequences of the exposure of tumor cells to selenium, we evaluated the selenium-dependent responses of two selenoproteins (glutathione peroxidase and the recently characterized 15-kDa selenoprotein) in three human glioma cell lines. Protein levels, mRNA levels, and the relative distribution of the two selenocysteine tRNA isoacceptors (designated mcm(5)U and mcm(5)Um) were determined for standard as well as selenium-supplemented conditions. The human malignant glioma cell lines D54, U251, and U87 were maintained in normal or selenium-supplemented (30 nM sodium selenite) conditions.

Inhibition of selenoprotein synthesis by selenocysteine tRNA[Ser]Sec lacking isopentenyladenosine.

A common posttranscriptional modification of tRNA is the isopentenylation of adenosine at position 37, creating isopentenyladenosine (i(6)A). The role of this modified nucleoside in protein synthesis of higher eukaryotes is not well understood. Selenocysteyl (Sec) tRNA (tRNA([Ser]Sec)) decodes specific UGA codons and contains i(6)A.

Yeast asparagine (Asn) tRNA without Q base promotes eukaryotic frameshifting more efficiently than mammalian Asn tRNAs with or without Q base.

In this study, we compare the efficiency of Asn tRNA from mammalian sources with and without the highly modified queuosine (Q) base in the wobble position of its anticodon and Asn tRNA from yeast, which naturally lacks Q base, to promote frameshifting. Interestingly, no differences in the ability of the two mammalian Asn tRNAs to promote frameshifting were observed, while yeast tRNA(ASn)(-Q) promoted frameshifting more efficiently than its mammalian counterparts in both rabbit reticulocyte lysates and wheat germ extracts. The shiftability of yeast Asn tRNA is therefore not due, or at least not completely, to the lack of Q base and most likely the shiftiness resides in structural differences elsewhere in the molecule.

A novel RNA binding protein, SBP2, is required for the translation of mammalian selenoprotein mRNAs.

In eukaryotes, the decoding of the UGA codon as selenocysteine (Sec) requires a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. We purified a SECIS binding protein, SBP2, and obtained a cDNA clone that encodes this activity. SBP2 is a novel protein containing a putative RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination.

Analysis of selenocysteine (Sec) tRNA([Ser]Sec) genes in Chinese hamsters.

Several recent observations have indicated that the primary structure of the Chinese hamster selenocysteine tRNA([Ser]sec) is different than those of other mammalian species. These reports prompted us to investigate the gene sequence for this tRNA in Chinese hamsters. Southern blotting of Chinese hamster ovary (CHO) genomic DNA derived from cultured cells with a tRNA([Ser]sec) probe indicated several hybridizing bands, and each of the corresponding genetic loci was isolated from a recombinant CHO library by molecular cloning.

Directed molecular evolution.

We propose the existence of a relationship of stereochemical complementarity between gene sequences that code for interacting components: nucleic acid-nucleic acid, protein-protein and protein-nucleic acid. Such a relationship would impose evolutionary constraints on the DNA sequences themselves, thus retaining these sequences and governing the direction of the evolutionary process. Therefore, we propose that prebiotic, template-directed autocatalytic synthesis of mutally cognate peptides and polynucleotides resulted in their amplification and evolutionary conservation in contemporary prokaryotic and eukaryotic organisms as a genetic regulatory apparatus.

Redox regulation of cell signaling by selenocysteine in mammalian thioredoxin reductases.

The intracellular generation of reactive oxygen species, together with the thioredoxin and glutathione systems, is thought to participate in redox signaling in mammalian cells. The activity of thioredoxin is dependent on the redox status of thioredoxin reductase (TR), the activity of which in turn is dependent on a selenocysteine residue. Two mammalian TR isozymes (TR2 and TR3), in addition to that previously characterized (TR1), have now been identified in humans and mice.

The zebrafish genome contains two distinct selenocysteine tRNA[Ser]sec genes.

The zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome.

Selenium metabolism in Drosophila. Characterization of the selenocysteine tRNA population.

The selenocysteine (Sec) tRNA population in Drosophila melanogaster is aminoacylated with serine, forms selenocysteyl-tRNA, and decodes UGA. The Km of Sec tRNA and serine tRNA for seryl-tRNA synthetase is 6.67 and 9.

Selenocysteine-containing thioredoxin reductase in C. elegans.

Mammalian thioredoxin reductases contain a TGA-encoded C-terminal penultimate selenocysteine (Sec) residue, and show little homology to bacterial, yeast, and plant thioredoxin reductases. Here we show that the nematode, Caenorhabditis elegans, contains two homologs related to the mammalian thioredoxin reductase family. The gene for one of these homologs contains a cysteine codon in place of TGA, and its product, designated TR-S, was previously suggested to function as thioredoxin reductase.

Selenocysteine-containing proteins in mammals.

Since the recent discovery of selenocysteine as the 21st amino acid in protein, the field of selenium biology has rapidly expanded. Twelve mammalian selenoproteins have been characterized to date and each contains selenocysteine that is incorporated in response to specific UGA code words. These selenoproteins have different cellular functions, but in those selenoproteins for which the function is known, selenocysteine is located at the active center.

Recruitment of TBP or TFIIB to a promoter proximal position leads to stimulation of RNA polymerase II transcription without activator proteins both in vivo and in vitro.

Eukaryotic transcriptional activators may function, at least in part, to facilitate the assembly of the RNA polymerase II (pol II) preinitiation complex at the core promoter region through their interaction with a subset of components of the basal transcription machinery. Previous studies have shown that artificial tethering of TATA-binding protein (TBP) to the promoter region is sufficient to stimulate pol II transcription in yeast. To test whether this phenomenon is a general one in eukaryotic pol II transcription, the DNA-binding domain of yeast GAL4 was fused to either Xenopus laevis TBP or TFIIB in order to enable these factors to be efficiently positioned near the transcription start site in a GAL4-binding site-dependent manner.

Transfer RNA modification status influences retroviral ribosomal frameshifting.

The possibility of whether tRNAs with and without a highly modified base in their anticodon loop may influence the level of retroviral ribosomal frameshifting was examined. Rabbit reticulocyte lysates were programmed with mRNA encoding UUU or AAC at the frameshift site and the corresponding Phe tRNA with or without the highly modified wyebutoxine (Y) base on the 3' side of its anticodon or Asn tRNA with or without the highly modified queuine (Q) base in the wobble position of its anticodon added. Phe and Asn tRNAs without the Y or Q base, respectively, stimulated the level of frameshifting, suggesting that the frameshift event is influenced by tRNA modification status.

Levels of major selenoproteins in T cells decrease during HIV infection and low molecular mass selenium compounds increase.

It has been observed previously that plasma selenium and glutathione levels are subnormal in HIV-infected individuals, and plasma glutathione peroxidase activity is decreased. Under these conditions the survival rate of AIDS patients is reduced significantly. In the present study, using 75Se-labeled human Jurkat T cells, we show that the levels of four 75Se-containing proteins are lower in HIV-infected cell populations than in uninfected cells.