tRNA Sec Gene Research Articles

Bioinformatic Prediction of an tRNASec Gene Nested inside an Elongation Factor SelB Gene in Alphaproteobacteria

In bacteria, selenocysteine (Sec) is incorporated into proteins via the recoding of a particular codon, the UGA stop codon in most cases. Sec-tRNASec is delivered to the ribosome by the Sec-dedicated elongation factor SelB that also recognizes a Sec-insertion sequence element following the codon on the mRNA. Since the excess of SelB may lead to sequestration of Sec-tRNASec under selenium deficiency or oxidative stress, the expression levels of SelB and tRNASec should be regulated. In this bioinformatic study, I analyzed the Rhizobiales SelB species because they were annotated to have a non-canonical C-terminal extension. I found that the open reading frame (ORF) of diverse Alphaproteobacteria selB genes includes an entire tRNASec sequence (selC) and overlaps with the start codon of the downstream ORF. A remnant tRNASec sequence was found in the Sinorhizobium meliloti selB genes whose products have a shorter C-terminal extension. Similar overlapping traits were found in Gammaproteobacteria and Nitrospirae. I hypothesized that once the tRNASec moiety is folded and processed, the expression of the full-length SelB may be repressed. This is the first report on a nested tRNA gene inside a protein ORF in bacteria.

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.

Selective Up-regulation of Human Selenoproteins in Response to Oxidative Stress

Selenocysteine is inserted into selenoproteins via the translational recoding of a UGA codon, normally used as a stop signal. This process depends on the nature of the selenocysteine insertion sequence element located in the 3' UTR of selenoprotein mRNAs, selenium bioavailability, and, possibly, exogenous stimuli. To further understand the function and regulation of selenoproteins in antioxidant defense and redox homeostasis, we investigated how oxidative stress influences selenoprotein expression as a function of different selenium concentrations. We found that selenium supplementation of the culture media, which resulted in a hierarchical up-regulation of selenoproteins, protected HEK293 cells from reactive oxygen species formation. Furthermore, in response to oxidative stress, we identified a selective up-regulation of several selenoproteins involved in antioxidant defense (Gpx1, Gpx4, TR1, SelS, SelK, and Sps2). Interestingly, the response was more efficient when selenium was limiting. Although a modest change in mRNA levels was noted, we identified a novel translational control mechanism stimulated by oxidative stress that is characterized by up-regulation of UGA-selenocysteine recoding efficiency and relocalization of SBP2, selenocysteine-specific elongation factor, and L30 recoding factors from the cytoplasm to the nucleus.

Selenoproteins Are Essential for Proper Keratinocyte Function and Skin Development

Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec). Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp) ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14) expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.

Mouse Models Targeting Selenocysteine tRNA Expression for Elucidating the Role of Selenoproteins in Health and Development

Selenium (Se) deficiency has been known for many years to be associated with disease, impaired growth and a variety of other metabolic disorders in mammals. Only recently has the major role that Se-containing proteins, designated selenoproteins, play in many aspects of health and development begun to emerge. Se is incorporated into protein by way of the Se-containing amino acid, selenocysteine (Sec). The synthesis of selenoproteins is dependent on Sec tRNA for insertion of Sec, the 21st amino acid in the genetic code, into protein. We have taken advantage of this dependency to modulate the expression of Sec tRNA that in turn modulates the expression of selenoproteins by generating transgenic, conditional knockout, transgenic/standard knockout and transgenic/conditional knockout mouse models, all of which involve the Sec tRNA gene, to elucidate the intracellular roles of this protein class.

The selenocysteine tRNA STAF-binding region is essential for adequate selenocysteine tRNA status, selenoprotein expression and early age survival of mice

STAF [Sec (selenocysteine) tRNA gene transcription activating factor] is a transcription activating factor for a number of RNA Pol III- and RNA Pol II-dependent genes including the Trsp [Sec tRNA gene], which in turn controls the expression of all selenoproteins. Here, the role of STAF in regulating expression of Sec tRNA and selenoproteins was examined. We generated transgenic mice expressing the Trsp transgene lacking the STAF-binding site and made these mice dependent on the transgene for survival by removing the wild-type Trsp. The level of Sec tRNA was unaffected or slightly elevated in heart and testis, but reduced approximately 60% in liver and kidney, approximately 70% in lung and spleen and approximately 80% in brain and muscle compared with the corresponding organs in control mice. Moreover, the ratio of the two isoforms of Sec tRNA that differ by methylation at position 34 (Um34) was altered significantly, and the Um34-containing form was substantially reduced in all tissues examined. Selenoprotein expression in these animals was most affected in tissues in which the Sec tRNA levels were most severely reduced. Importantly, mice had a neurological phenotype strikingly similar to that of mice in which the selenoprotein P gene had been removed and their life span was substantially reduced. The results indicate that STAF influences selenoprotein expression by enhancing Trsp synthesis in an organ-specific manner and by controlling Sec tRNA modification in each tissue examined.

Targeted removal of the selenocysteine tRNA gene (trsp) in epidermis modulates skin function and development.

Selenium is an essential dietary micronutrient having numerous health benefits. It is incorporated into proteins (selenoproteins) as selenocysteine (Sec). Selenoprotein synthesis depends on Sec tRNA, a unique eukaryotic tRNA that governs the expression of only a single class of proteins, the selenoproteins. Manipulating Sec tRNA structure and expression can selectively alter selenoprotein synthesis. To understand the importance of selenoprotein expression in skin development and function, we targeted the removal of the Sec tRNA gene (trsp) in mouse epidermis, using mammalian K14 promoter based loxP‐Cre technology. Our results show that the knockout mice (Δtrsp) have stunted growth and a shorter life span as compared to control mice. They have flaky and wrinkled skin and histological analysis revealed moderate epidermal hyperplasia along with coagulative necrosis of the epidermis. The knockout mice have sparse hair and histological examination suggested that hair morphogenesis might be affected. Real‐time PCR analysis of skin of knockout and control mice shows a decrease in the expression of selenoprotein mRNAs in knockout mice, with the decrease being most significant in epidermis. These studies report a novel and previously unidentified role of selenoproteins in skin function and development. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.

The selenocysteine tRNA Staf region is essential for adequate selenoprotein levels and mouse longevity

Selenocysteine (Sec) tRNA has been described as the key molecule and the central component in selenoprotein (SP) biosynthesis and is the only known tRNA that governs the expression of an entire class of proteins, the SPs. This provides a unique opportunity to study the expression of SPs by manipulating the levels and characteristics of Sec tRNA. A cDNA encoding a protein that binds to the activator element of Sec tRNA was cloned from X. laevis and named Staf (for Sec tRNA gene transcription activating factor) (Adachi, et al. J Biol Chem 273:, 1998). Herein, we generated a transgenic mouse harboring multiple copies of a transgene carrying a 14bp deletion in the Staf binding region of the Sec tRNA gene (trsp), which is located approximately −200bp upstream of trsp. Transgenic mice were bred with a trsp knockout mouse to remove endogenous wild type trsp. Mice dependent on the mutant transgene showed ~80% decrease in the levels of Sec tRNA in liver. Se75-labeling and western blot analysis revealed that several SPs were affected by this mutation to varying degrees depending on tissue type. The mutant mice showed decreased size and body weight and had a lifespan of less than 30 days. Thus, the Staf binding region of trsp is essential for proper amounts of Sec tRNA transcription. These and other recent findings will be presented. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.

A comparative analysis of selenoproteins and global gene expression in liver selenocysteine tRNA knockout mice and its rescued variants

Selenium (Se) is an essential micronutrient for mammals, being incorporated into selenoproteins (SPs) as the 21st amino acid, selenocysteine (Sec). Rodents and humans encode 24 and 25 SP genes, respectively. SPs are tissue specific, performing biologically diverse functions. Sec expression is dependent on Sec tRNA, thus, manipulating the expression of Sec tRNA can alter SP expression. Targeted removal of the Sec tRNA gene (trsp) in liver demonstrated the significance of SPs in liver function (Carlson et al., J. Biol. Chem., 2004, 279:). The SP population was replaced in these mice with mutant variants of trsp (A34 and G37) and microarrays were used for comparative analysis of gene expression in liver of knockout and rescued mice. The SPs in these mice were further assessed through real-time PCR. Studies revealed a loss of expression of most SPs in the knockout mice with an enhanced expression of some detoxification genes. However, in the replacement mice, most SPs were expressed similar to the wild type with a corresponding decrease in the expression of detoxification genes as compared to the knockout. Taken together, the data suggest a relationship between SPs and detoxification processes, wherein some specific SPs may be compensated for by the upregulation of genes involved in detoxification in liver of the knockout mouse. This research was supported by the Intramural Research Program of the NIH, NCI, CCR, and by NIH GM061603.

Selective Rescue of Selenoprotein Expression in Mice Lacking a Highly Specialized Methyl Group in Selenocysteine tRNA

Selenocysteine (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.

Models for Assessing the Role of Selenoproteins in Health

Two model systems for examining the role of selenoproteins in health are discussed. One utilizes transgenic mice that carry mutant selenocysteine (Sec) tRNA transgenes that result in the reduction of selenoprotein expression in a protein- and tissue-specific manner. The other utilizes loxP-Cre technology to selectively remove the Sec tRNA gene in mammary epithelium that results in the reduction of only certain selenoproteins in this tissue. Both approaches provide important tools for examining the role of selenoproteins in health.

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 9.45 nM, respectively. Two major bands of Sec tRNA were resolved by gel electrophoresis. Both tRNAs were sequenced, and their primary structures were indistinguishable and colinear with that of the corresponding single copy gene. They are 90 nucleotides in length and contain three modified nucleosides, 5-methylcarboxymethyluridine, N6-isopentenyladenosine, and pseudouridine, at positions 34, 37, and 55, respectively. Neither form contains 1-methyladenosine at position 58 or 5-methylcarboxymethyl-2'-O-methyluridine, which are characteristically found in Sec tRNA of higher animals. We conclude that the primary structures of the two bands of Sec tRNA resolved by electrophoresis are indistinguishable by the techniques employed and that Sec tRNAs in Drosophila may exist in different conformational forms. The Sec tRNA gene maps to a single locus on chromosome 2 at position 47E or F. To our knowledge, Drosophila is the lowest eukaryote in which the Sec tRNA population has been characterized to date.

Molecular Cloning and Characterization of the Murine Staf cDNA Encoding a Transcription Activating Factor for the Selenocysteine tRNA Gene in Mouse Mammary Gland

We have isolated and characterized a cDNA encoding a transcription activating factor for the mouse selenocysteine tRNA (tRNAsec) gene from mouse mammary gland. The full-length cDNA, designated m-Staf, has a 1878-base pair open reading frame encoding 626 amino acids. The predicted amino acid sequence of m-Staf is highly homologous to that of Staf, another selenocysteine tRNA gene transcription activating factor of Xenopus laevis. Like Staf, m-Staf contains seven tandemly repeated zinc fingers and four repeated motifs. Gel shift assays indicated that the recombinant m-Staf specifically bound to the activator element region in the mouse tRNAsec gene. Transient co-transfection experiments in Drosophila Schneider cells, which lack endogenous Staf-like binding activity, showed that m-Staf increased the mouse tRNAsec gene transcription about 15-fold, whereas it stimulated Pol II-dependent thymidine kinase promoter only 2-fold. Northern blot analysis detected the presence of a 3.4-kilobase pair m-Staf transcript, which was widely but differentially expressed in various murine tissues. The binding activity of m-Staf in mouse mammary gland was undetectable during virgin and postlactating periods but increased markedly in parallel with the increase of tRNAsec transcript during the periods of pregnancy and lactation, when the gland undergoes growth and development. These results indicate that m-Staf is a transcriptional activator of the mouse tRNAsec gene and that its binding activity in the mammary gland undergoes developmental alterations.

Native bovine selenocysteine tRNA sec secondary structure as probed by two plant single-strand-specific nucleases

Two single-strand-specific nucleases, discovered in plants, have been used to investigate the secondary and tertiary structures of the native bovine liver selenocysteine tRNA Sec. To check the possible influence of nucleotide modifications on these structures, we compared the results obtained with the fully modified tRNA to the unmodified transcript prepared by in vitro T7 transcription of the Xenopus laevis tRNA Sec gene. We found that the structures in solution of the native tRNA Sec and the transcript are very similar despite some differences in accessibility to the enzymatic probes. Indeed, the modified anticodon-loop of native bovine tRNA Sec, containing 5-methylcarboxymethyluridine (mcm 5U 34) and N6-isopentenyladenosine (i6A37), is less accessible to Rn nuclease than that of the transcript: the intensity of bands representing cuts at A 36 and A 38 is much lower as compared to those of the transcript, whereas no cuts were found at the level of i 6A 37 in the anticodon loop of the native molecule. Surprisingly, the variable arm of the native molecule has been found to be more susceptible to single-strand-specific nuclease action, suggesting a looser structure of the variable arm in native bovine tRNA sec than in the transcript.

Eukaryotic selenocysteine inserting tRNA species support selenoprotein synthesis in Escherichia coli.

Although the tRNA species directing selenocysteine insertion in prokaryotes differ greatly in their primary structure from that of their eukaryotic homologues they share very similar three-dimensional structures. To analyse whether this conservation of the overall shape of the molecules reflects a conservation of their functional interactions it was tested whether the selenocysteine inserting tRNA species from Homo sapiens supports selenoprotein synthesis in E. coli. It was found that the expression of the human tRNA(Sec) gene in E.coli can complement a lesion in the tRNA(Sec) gene of this organism. Transcripts of the Homo sapiens and Xenopus laevis tRNA(Sec) genes synthesised in vitro were amino-acylated by the E.coli seryl-tRNA ligase although at a very low rate and the resulting seryl-tRNA(Sec) was bound to and converted into selenocysteyl-tRNA(Sec) by the selenocysteine synthase of this organism. Selenocysteyl-tRNA(Sec) from both eukaryotes was able to form a complex with translation factor SELB from E.coli. Although the mechanism of selenocysteine incorporation into seleno-proteins appears to be rather different in E.coli and in vertebrates, we observe here a surprising conservation of functions over an enormous evolutionary distance.

Promoter Strength and Structure Dictate Module Composition in RNA Polymerase III Transcriptional Activator Elements

RNA polymerase III transcription of genes with external promoters only (e.g. U6 snRNA) or containing in addition an internal B box (selenocysteine tRNA Sec) is stimulated by upstream elements; a distal sequence element (DSE) for U6 or an activator element in the tRNA Sec gene. In contrast to the composite structure of the DSE which requires an octamer motif, the Xenopus tRNA Sec activator element contains an SPH motif only. In vivo transcription is optimally stimulated by SPH in an absolute octamer-independent manner since adding octamer does not induce superstimulation. Experiments performed in the work presented here led to the following observations. Co-operation between SPH and octamer motifs can be detected in two distinct cases: first when these motifs are placed in front of B box-less tRNA Sec or U6 external promoters and second, if either element of the external promoter (proximal sequence element or TATA element), or the SPH motif itself, are altered. Altogether, our data provide evidence that an SPH motif can function alone in an optimized promoter only. In contrast, an octamer becomes indispensable when the basal promoter is weak or disabled. It follows that module composition of Pol III transcriptional activator elements is dependent on the structure and strength of the promoter. This reveals the existence of cross-talk between activator and promoter elements, mediated by the bound transcription factors, which are thus able to compensate for each other in order to allow successful assembly of the transcription complex.