Molecular mechanism of selenoprotein P synthesis

Background

Selenoprotein synthesis requires the reinterpretation of a UGA stop codon as one that encodes selenocysteine (Sec), a process that requires a set of dedicated translation factors. Among the mammalian selenoproteins, Selenoprotein P (SELENOP) is unique as it contains a selenocysteine-rich domain that requires multiple Sec incorporation events.

Selenium-regulated hierarchy of human selenoproteome in cancerous and immortalized cells lines

Background

Selenoproteins (25 genes in human) co-translationally incorporate selenocysteine using a UGA codon, normally used as a stop signal. The human selenoproteome is primarily regulated by selenium bioavailability with a tissue-specific hierarchy.

On elongation factor eEFSec,its role and mechanism during selenium incorporation into nascent selenoproteins

Background

Selenium, an essential dietary micronutrient, is incorporated into proteins as the amino acid selenocysteine (Sec) in response to in-frame UGA codons. Complex machinery ensures accurate recoding of Sec codons in higher organisms. A specialized elongation factor eEFSec is central to the process.

Selenoprotein synthesis and regulation in Archaea

Background

The major biological form of selenium is that of the co-translationally inserted amino acid selenocysteine (Sec). In Archaea, the majority of proteins containing Sec, selenoproteins, are involved in methanogenesis. However, the function of this residue is often not known because selenium-independent homologs of the selenoproteins can be employed, sometimes even in one organism.

Delivery of selenium to selenophosphate synthetase for selenoprotein biosynthesis

Background

Selenophosphate, the key selenium donor for the synthesis of selenoprotein and selenium-modified tRNA, is produced by selenophosphate synthetase (SPS) from ATP, selenide, and H₂O. Although free selenide can be used as the in vitro selenium substrate for selenophosphate synthesis, the precise physiological system that donates in vivo selenium substrate to SPS has not yet been characterized completely.

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.     

Evolutionary history of selenocysteine incorporation from the perspective of SECIS binding proteins

Background  

The co-translational incorporation of selenocysteine into nascent polypeptides by recoding the UGA stop codon occurs in all domains of life. In eukaryotes, this event requires at least three specific factors: SECIS binding protein 2 (SBP2), a specific translation elongation factor (eEFSec), selenocysteinyl tRNA, and a cis -acting selenocysteine insertion sequence (SECIS) element in selenoprotein mRNAs. While the phylogenetic relationships of selenoprotein families and the evolution of selenocysteine usage are well documented, the evolutionary history of SECIS binding proteins has not been explored.     

Translational Redefinition of UGA Codons Is Regulated by Selenium Availability

Incorporation of selenium into ∼25 mammalian selenoproteins occurs by translational recoding whereby in-frame UGA codons are redefined to encode the selenium containing amino acid, selenocysteine (Sec). Here we applied ribosome profiling to examine the effect of dietary selenium levels on the translational mechanisms controlling selenoprotein synthesis in mouse liver. Dietary selenium levels were shown to control gene-specific selenoprotein expression primarily at the translation level by differential regulation of UGA redefinition and Sec incorporation efficiency, although effects on translation initiation and mRNA abundance were also observed. Direct evidence is presented that increasing dietary selenium causes a vast increase in ribosome density downstream of UGA-Sec codons for a subset of selenoprotein mRNAs and that the selenium-dependent effects on Sec incorporation efficiency are mediated in part by the degree of Sec-tRNA^[Ser]Sec Um34 methylation. Furthermore, we find evidence for translation in the 5′-UTRs for a subset of selenoproteins and for ribosome pausing near the UGA-Sec codon in those mRNAs encoding the selenoproteins most affected by selenium availability. These data illustrate how dietary levels of the trace element selenium can alter the readout of the genetic code to affect the expression of an entire class of proteins.     

Regulation and function of avian selenogenome

Background

Selenium (Se) is an essential micronutrient required by avian species. Dietary Se/vitamin E deficiency induces three classical diseases in chicks: exudative diathesis, nutritional pancreatic atrophy, and nutritional muscular dystrophy.

A tale of two toxicities: malformed selenoproteins and oxidative stress both contribute to selenium stress in plants

Background

Despite selenium''s toxicity in plants at higher levels, crops supply most of the essential dietary selenium in humans. In plants, inorganic selenium can be assimilated into selenocysteine, which can replace cysteine in proteins. Selenium toxicity in plants has been attributed to the formation of non-specific selenoproteins. However, this paradigm can be challenged now that there is increasingly abundant evidence suggesting that selenium-induced oxidative stress also contributes to toxicity in plants.

Scope

This Botanical Briefing summarizes the evidence indicating that selenium toxicity in plants is attributable to both the accumulation of non-specific selenoproteins and selenium-induced oxidative stress. Evidence is also presented to substantiate the claim that inadvertent selenocysteine replacement probably impairs or misfolds proteins, which supports the malformed selenoprotein hypothesis. The possible physiological ramifications of selenoproteins and selenium-induced oxidative stress are discussed.

Conclusions

Malformed selenoproteins and oxidative stress are two distinct types of stress that drive selenium toxicity in plants and could impact cellular processes in plants that have yet to be thoroughly explored. Although challenging, deciphering whether the extent of selenium toxicity in plants is imparted by selenoproteins or oxidative stress could be helpful in the development of crops with fortified levels of selenium.     

Processive Selenocysteine Incorporation during Synthesis of Eukaryotic Selenoproteins

Selenoproteins are a family of proteins that share the common feature of containing selenocysteine, the “twenty-first” amino acid. Selenocysteine incorporation occurs during translation of selenoprotein messages by redefinition of UGA codons, which normally specify termination of translation. Studies of the eukaryotic selenocysteine incorporation mechanism suggest that selenocysteine insertion is inefficient compared with termination. Nevertheless, selenoprotein P and several other selenoproteins are known to contain multiple selenocysteines. The production of full-length (FL) protein from these messages would seem to demand highly efficient selenocysteine incorporation due to the compounding effect of termination at each UGA codon. We present data demonstrating that efficient incorporation of multiple selenocysteines can be reconstituted in rabbit reticulocyte lysate translation reactions. Selenocysteine incorporation at the first UGA codon is inefficient but increases by approximately 10-fold at subsequent downstream UGA codons. We found that ribosomes in the “processive” phase of selenocysteine incorporation (i.e., after decoding the first UGA codon as selenocysteine) are fully competent to terminate translation at UAG and UAA codons, that ribosomes become less efficient at selenocysteine incorporation as the distance between UGA codons is increased, and that efficient selenocysteine incorporation is not dependent on cis-acting elements unique to selenoprotein P. Furthermore, we found that the percentage of ribosomes decoding a UGA codon as selenocysteine rather than termination can be increased by 3- to 5-fold by placing the murine leukemia virus UAG read-through element upstream of the first UGA codon or by providing a competing messenger RNA in trans. The mechanisms of selenocysteine incorporation and selenoprotein synthesis are discussed in light of these results.     

Selenium unmasks protective iron armor: A possible defense against cutaneous inflammation and cancer

Background

A link between selenium deficiency and inflammatory skin diseases have been noted by many, but this link is still not well understood. We have previously studied the efficacy of ceramide analogs, based on the fire ant venom Solenopsin A, against our psoriasis animal model. Treatment of animals with solenopsin analogs resulted in significantly improved skin as well as in a coordinate downregulation of selenoproteins, namely Glutathione Peroxidase 4 (GPX4). We thus hypothesize that ferroptosis may be a physiologic process that may protect the skin from both inflammatory and neoplastic processes.

Genetic polymorphism in selenoprotein P modifies the response to selenium-rich foods on blood levels of selenium and selenoprotein P in a randomized dietary intervention study in Danes

Background

Selenium is an essential trace element and is suggested to play a role in the etiology of a number of chronic diseases. Genetic variation in genes encoding selenoproteins, such as selenoprotein P and the glutathione peroxidases, may affect selenium status and, thus, individual susceptibility to some chronic diseases. In the present study, we aimed to (1) investigate the effect of mussel and fish intake on glutathione peroxidase enzyme activity and (2) examine whether single nucleotide polymorphisms in the GPX1, GPX4, and SELENOP genes modify the effect of mussel and fish intake for 26 weeks on whole blood selenium, plasma selenoprotein P concentrations, and erythrocyte GPX enzyme activity in a randomized intervention trial in Denmark.

Results

CC homozygotes of the SELENOP/rs3877899 polymorphism who consumed 1000 g fish and mussels per week for 26 consecutive weeks had higher levels of both selenoprotein P (difference between means ??4.68 ng/mL (95% CI ??8.49, ??0.871)) and whole blood selenium (difference between means ??5.76 (95% CI ??12.5, 1.01)) compared to fish and mussel consuming T-allele carriers although the effect in whole blood selenium concentration was not statistically significant.

Conclusions

Our study indicates that genetically determined variation in SELENOP leads to different responses in expression of selenoproteins following consumption of selenium-rich foods. This study also emphasizes the importance of taking individual aspects such as genotypes into consideration when assessing risk in public health recommendations.

     

Partitioning between recoding and termination at a stop codon–selenocysteine insertion sequence

Selenocysteine (Sec) is inserted into proteins by recoding a UGA stop codon followed by a selenocysteine insertion sequence (SECIS). UGA recoding by the Sec machinery is believed to be very inefficient owing to RF2-mediated termination at UGA. Here we show that recoding efficiency in vivo is 30–40% independently of the cell growth rate. Efficient recoding requires sufficient selenium concentrations in the medium. RF2 is an unexpectedly poor competitor of Sec. We recapitulate the major characteristics of SECIS-dependent UGA recoding in vitro using a fragment of fdhF-mRNA encoding a natural bacterial selenoprotein. Only 40% of actively translating ribosomes that reach the UGA codon insert Sec, even in the absence of RF2, suggesting that the capacity to insert Sec into proteins is inherently limited. RF2 does not compete with the Sec incorporation machinery; rather, it terminates translation on those ribosomes that failed to incorporate Sec. The data suggest a model in which early recruitment of Sec-tRNA^Sec–SelB–GTP to the SECIS blocks the access of RF2 to the stop codon, thereby prioritizing recoding over termination at Sec-dedicated stop codons.     

A protein binds the selenocysteine insertion element in the 3'-UTR of mammalian selenoprotein mRNAs.

Several gene products are involved in co-translational insertion of selenocysteine by the tRNA(Sec). In addition, a stem-loop structure in the mRNAs coding for selenoproteins is essential to mediate the selection of the proper selenocysteine UGA codon. Interestingly, in eukaryotic selenoprotein mRNAs, this stem-loop structure, the selenocysteine insertion sequence (SECIS) element, resides in the 3'-untranslated region, far downstream of the UGA codon. In view of unravelling the underlying complex mechanism, we have attempted to detect RNA-binding proteins with specificity for the SECIS element. Using mobility shift assays, we could show that a protein, present in different types of mammalian cell extracts, possesses the capacity of binding the SECIS element of the selenoprotein glutathione peroxidase (GPx) mRNA. We have termed this protein SBP, for Secis Binding Protein. Competition experiments attested that the binding is highly specific and UV cross-linking indicated that the protein has an apparent molecular weight in the range of 60-65 kDa. Finally, some data suggest that the SECIS elements in the mRNAs of GPx and another selenoprotein, type I iodothyronine 5' deiodinase, recognize the same SBP protein. This constitutes the first report of the existence of a 3' UTR binding protein possibly involved in the eukaryotic selenocysteine insertion mechanism.     

Efficient incorporation of multiple selenocysteines involves an inefficient decoding step serving as a potential translational checkpoint and ribosome bottleneck

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.     

SECIS elements in the coding regions of selenoprotein transcripts are functional in higher eukaryotes

Expression of selenocysteine (Sec)-containing proteins requires the presence of a cis-acting mRNA structure, called selenocysteine insertion sequence (SECIS) element. In bacteria, this structure is located in the coding region immediately downstream of the Sec-encoding UGA codon, whereas in eukaryotes a completely different SECIS element has evolved in the 3'-untranslated region. Here, we report that SECIS elements in the coding regions of selenoprotein mRNAs support Sec insertion in higher eukaryotes. Comprehensive computational analysis of all available viral genomes revealed a SECIS element within the ORF of a naturally occurring selenoprotein homolog of glutathione peroxidase 4 in fowlpox virus. The fowlpox SECIS element supported Sec insertion when expressed in mammalian cells as part of the coding region of viral or mammalian selenoproteins. In addition, readthrough at UGA was observed when the viral SECIS element was located upstream of the Sec codon. We also demonstrate successful de novo design of a functional SECIS element in the coding region of a mammalian selenoprotein. Our data provide evidence that the location of the SECIS element in the untranslated region is not a functional necessity but rather is an evolutionary adaptation to enable a more efficient synthesis of selenoproteins.