Bradley A. Carlson

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. By UV cross‐linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using 75Se‐labeled Sec‐tRNASec, we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co‐translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.

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. We have identified and characterized the first eukaryotic selenocysteyl‐tRNA‐specific elongation factor. This factor forms a complex with mammalian SECIS binding protein 2, and these two components function together in selenocysteine incorporation in mammalian cells. Expression of the two functional domains of the bacterial elongation factor–SECIS binding protein as two separate proteins in eukaryotes suggests a mechanism for rapid exchange of charged for uncharged selenocysteyl‐tRNA–elongation factor complex, allowing a single SECIS element to serve multiple UGA codons.

Thioredoxin Reductase 1 Deficiency Reverses Tumor Phenotype and Tumorigenicity of Lung Carcinoma Cells

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.

Selenium and selenocysteine: roles in cancer, health, and development

The many biological and biomedical effects of selenium are relatively unknown outside the selenium field. This fascinating element, initially described as a toxin, was subsequently shown to be essential for health and development. By the mid-1990s selenium emerged as one of the most promising cancer chemopreventive agents, but subsequent human clinical trials yielded contradictory results. However, basic research on selenium continued to move at a rapid pace, elucidating its many roles in health, development, and in cancer prevention and promotion. Dietary selenium acts principally through selenoproteins, most of which are oxidoreductases involved in diverse cellular functions.

The kinase p38|[alpha]| serves cell type|[ndash]|specific inflammatory functions in skin injury and coordinates pro- and anti-inflammatory gene expression

The mitogen-activated protein kinase p38 mediates cellular responses to injurious stress and immune signaling. Among the many p38 isoforms, p38α is the most widely expressed in adult tissues and can be targeted by various pharmacological inhibitors. Here we investigated how p38α activation is linked to cell type–specific outputs in mouse models of cutaneous inflammation. We found that both myeloid and epithelial p38α elicit inflammatory responses, yet p38α signaling in each cell type served distinct inflammatory functions and varied depending on the mode of skin irritation. In addition, myeloid p38α limited acute inflammation via activation of anti-inflammatory gene expression dependent on mitogen- and stress-activated kinases. Our results suggest a dual function for p38α in the regulation of inflammation and show mixed potential for its inhibition as a therapeutic strategy.

Hepatically derived selenoprotein P is a key factor for kidney but not for brain selenium supply.

Liver-specific inactivation of Trsp, the gene for selenocysteine tRNA, removes SePP (selenoprotein P) from plasma, causing serum selenium levels to fall from 298 microg/l to 50 microg/l and kidney selenium to decrease to 36% of wild-type levels. Likewise, glutathione peroxidase activities decreased in plasma and kidney to 43% and 18% respectively of wild-type levels. This agrees nicely with data from SePP knockout mice, supporting a selenium transport role for hepatically expressed SePP. However, brain selenium levels remain unaffected and neurological defects do not occur in the liver-specific Trsp knockout mice, while SePP knockout mice suffer from neurological defects. This indicates that a transport function in plasma is exerted by hepatically derived SePP, while in brain SePP fulfils a second, hitherto unexpected, essential role.

Selenocysteine incorporation machinery and the role of selenoproteins in development and health.

Publisher Summary One of the major areas of emphasis has understood the role of selenium in health. Selenium is an essential micronutrient in the diet of mammals, and this element has numerous health benefits. It has roles in cancer and heart disease prevention, inhibiting viral expression, and delaying the progression of AIDS in HIV positive patients. Selenium has been reported to have roles in immune function, male reproduction, mammalian development, and slowing the aging process. This chapter discusses the means by which amino acid selenocysteine (Sec) is biosynthesized and incorporated into protein; the generation of mouse models for elucidating the role of selenoproteins in development and health; the identity and functions of selenoproteins; and the distribution and evolution of the amino acid Sec insertion machinery among eukaryotes. Sec is biosynthesized, unlike the common biosynthetic pathways of the other 20 protein amino acids, on its transfer RNA (tRNA). The machinery for inserting Sec into protein is novel and unique to this amino acid. It is apparent that tremendous effort has been expended in evolution for inserting selenium into protein in the form of Sec as discussed in the chapter.

Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration

Cerebral selenium (Se) deficiency is associated with neurological phenotypes including seizures and ataxia. We wanted to define whether neurons require selenoprotein expression and which selenoproteins are most important, and explore the possible pathomechanism. Therefore, we abrogated the expression of all selenoproteins in neurons by genetic inactivation of the tRNA[Ser]Sec gene. Cerebral expression of selenoproteins was significantly diminished in the mutants, and histological analysis revealed progressive neurodegeneration. Developing interneurons failed to specifically express parvalbumin (PV) in the mutants. Electrophysiological recordings, before overt cell death, showed normal excitatory transmission, but revealed spontaneous epileptiform activity consistent with seizures in the mutants. In developing cortical neuron cultures, the number of PV+ neurons was reduced on combined Se and vitamin E deprivation, while other markers, such as calretinin (CR) and GAD67, remained unaffected. Because of the synergism between Se and vitamin E, we analyzed mice lacking neuronal expression of the Se‐dependent enzyme glutathione peroxidase 4 (GPx4). Although the number of CR+ interneurons remained normal in Gpx4‐mutant mice, the number of PV+ interneurons was reduced. Since these mice similarly exhibit seizures and ataxia, we conclude that GPx4 is a selenoenzyme modulating interneuron function and PV expression. Cerebral SE deficiency may thus act via reduced GPx4 expression.—Wirth, E. K., Conrad, M., Winterer, J., Wozny, C., Carlson, B. A., Roth, S., Schmitz, D., Bornkamm, G. W., Coppola, V., Tessarollo, L., Schomburg, L., Köhrle, J., Hatfield, D. L., Schweizer, U. Neuronal selenoprotein expression is required for interneuron development and prevents seizures and neurodegeneration. FASEB J. 24, 844–852 (2010). www.fasebj.org

Supramolecular complexes mediate selenocysteine incorporation in vivo.

ABSTRACT 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.

Selective Inhibition of Selenocysteine tRNA Maturation and Selenoprotein Synthesis in Transgenic Mice Expressing Isopentenyladenosine-Deficient Selenocysteine tRNA

ABSTRACT 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. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i6A−) tRNA[Ser]Sec in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA[Ser]Sec population showed that expression of i6A− tRNA[Ser]Sec altered the distribution of the two major isoforms, whereby the maturation of tRNA[Ser]Sec by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i6A− tRNA[Ser]Sec and wild-type tRNA[Ser]Sec are regulated independently and that the amount of wild-type tRNA[Ser]Sec is determined, at least in part, by a feedback mechanism governed by the level of the tRNA[Ser]Sec population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i6A−tRNA[Ser]Sec transgenic mice will be useful in assessing the biological roles of selenoproteins.