Selenocysteine Insertion Sequence Research Articles

The Specific Elongation Factor to Selenocysteine Incorporation in Escherichia coli: Unique tRNASec Recognition and its Interactions

Several molecular mechanisms are involved in the genetic code interpretation during translation, as codon degeneration for the incorporation of rare amino acids. One mechanism that stands out is selenocysteine (Sec), which requires a specific biosynthesis and incorporation pathway. In Bacteria, the Sec biosynthesis pathway has unique features compared with the eukaryote pathway as Ser to Sec conversion mechanism is accomplished by a homodecameric enzyme (selenocysteine synthase, SelA) followed by the action of an elongation factor (SelB) responsible for delivering the mature Sec-tRNASec into the ribosome by the interaction with the Selenocysteine Insertion Sequence (SECIS). Besides this mechanism being already described, the sequential events for Sec-tRNASec and SECIS specific recognition remain unclear. In this study, we determined the order of events of the interactions between the proteins and RNAs involved in Sec incorporation. Dissociation constants between SelB and the native as well as unacylated-tRNASec variants demonstrated that the acceptor stem and variable arm are essential for SelB recognition. Moreover, our data support the sequence of molecular events where GTP-activated SelB strongly interacts with SelA.tRNASec. Subsequently, SelB.GTP.tRNASec recognizes the mRNA SECIS to deliver the tRNASec to the ribosome. SelB in complex with its specific RNAs were examined using Hydrogen/Deuterium exchange mapping that allowed the determination of the molecular envelopes and its secondary structural variations during the complex assembly. Our results demonstrate the ordering of events in Sec incorporation and contribute to the full comprehension of the tRNASec role in the Sec amino acid biosynthesis, as well as extending the knowledge of synthetic biology and the expansion of the genetic code.

The Possible Mechanism of Physiological Adaptation to the Low-Se Diet and Its Health Risk in the Traditional Endemic Areas of Keshan Diseases.

Selenium is an essential trace element for humans and animals. As with oxygen and sulfur, etc., it belongs to the sixth main group of the periodic table of elements. Therefore, the corresponding amino acids, such as selenocysteine (Sec), serine (Ser), and cysteine (Cys), have similar spatial structure, physical, and chemical properties. In this review, we focus on the neglected but key role of serine in a possible mechanism of the physiological adaptation to Se-deficiency in human beings with an adequate intake of dietary protein: the insertion of Cys in place of Sec during the translation of selenoproteins dependent on the Sec insertion sequence element in the 3′UTR of mRNA at the UGA codon through a novel serine-dependent pathway for the de novo synthesis of the Cys-tRNA[Ser]Sec, similar to Sec-tRNA[Ser]Sec. We also discuss the important roles of serine in the metabolism of selenium directly or indirectly via GSH, and the maintenance of selenium homostasis regulated through the methylation modification of Sec-tRNA[Ser]Sec at the position 34U by SAM. Finally, we propose a hypothesis to explain why Keshan disease has gradually disappeared in China and predict the potential health risk of the human body in the physiological adaptation state of low selenium based on the results of animal experiments.

Identification of a novel endogenous long non-coding RNA that inhibits selenoprotein P translation.

Selenoprotein P (SELENOP) is a major plasma selenoprotein that contains 10 Sec residues, which is encoded by the UGA stop codon. The mRNA for SELENOP has the unique property of containing two Sec insertion sequence (SECIS) elements, which is located in the 3′ untranslated region (3′UTR). Here, we coincidentally identified a novel gene, CCDC152, by sequence analysis. This gene was located in the antisense region of the SELENOP gene, including the 3′UTR region in the genome. We demonstrated that this novel gene functioned as a long non-coding RNA (lncRNA) that decreased SELENOP protein levels via translational rather than transcriptional, regulation. We found that the CCDC152 RNA interacted specifically and directly with the SELENOP mRNA and inhibited its binding to the SECIS-binding protein 2, resulting in the decrease of ribosome binding. We termed this novel gene product lncRNA inhibitor of SELENOP translation (L-IST). Finally, we found that epigallocatechin gallate upregulated L-IST in vitro and in vivo, to suppress SELENOP protein levels. Here, we provide a new regulatory mechanism of SELENOP translation by an endogenous long antisense ncRNA.

Enhanced antioxidant capacity prevents epitranscriptomic and cardiac alterations in adult offspring gestationally-exposed to ENM

Maternal engineered nanomaterial (ENM) exposure during gestation has been associated with negative long-term effects on cardiovascular health in progeny. Here, we evaluate an epitranscriptomic mechanism that contributes to these chronic ramifications and whether overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) can preserve cardiovascular function and bioenergetics in offspring following gestational nano-titanium dioxide (TiO2) inhalation exposure. Wild-type (WT) and mPHGPx (Tg) dams were exposed to nano-TiO2 aerosols with a mass concentration of 12.01 ± 0.50 mg/m3 starting from gestational day (GD) 5 for 360 mins/day for 6 nonconsecutive days over 8 days. Echocardiography was performed in pregnant dams, adult (11-week old) and fetal (GD 14) progeny. Mitochondrial function and global N6-methyladenosine (m6A) content were assessed in adult progeny. MPHGPx enzymatic function was further evaluated in adult progeny and m6A-RNA immunoprecipitation (RIP) was combined with RT-qPCR to evaluate m6A content in the 3′-UTR. Following gestational ENM exposure, global longitudinal strain (GLS) was 32% lower in WT adult offspring of WT dams, with preservation in WT offspring of Tg dams. MPHGPx activity was significantly reduced in WT offspring (29%) of WT ENM-exposed dams, but preserved in the progeny of Tg dams. M6A-RIP-qPCR for the SEC insertion sequence region of mPHGPx revealed hypermethylation in WT offspring from ENM-exposed WT dams, which was thwarted in the presence of the maternal transgene. Our findings implicate that m6A hypermethylation of mPHGPx may be culpable for diminished antioxidant capacity and resultant mitochondrial and cardiac deficits that persist into adulthood following gestational ENM inhalation exposure.

High resolution ribosome profiling reveals gene‐specific details of UGA re‐coding in selenoproteins

SECIS binding protein 2 (SECISBP2) increases the efficiency of re-coding of UGA codons as selenocysteine (Sec) during translation of mRNAs containing a Sec insertion sequence (SECIS) in the 3’ untranslated region. Our previous ribosomal profiling (RiboSeq) studies in mice showed that re-coding of UGA occurred in the absence of Secisbp2 on a higher level than if tRNASec was inactivated. We concluded that UGA recoding was an intrinsic property of UGA codons in selenoproteins which can be massively increased by the SECIS: Secisbp2 interaction. Since we worked on cell-type specific liver and brain tissues, heterogeneity of cells expressing or not wildtype Secisbp2 might have confounded our analyses and conclusions. In order to study a homogenous sample and thus gain a more detailed in-depth view on UGA recoding, we decided to subject the haploid human HAP1 cell line deficient in SECISBP2 by RiboSeq. Expression of selenoproteins was found at a reduced, but still detectable, level in the mutated cells. Metabolic labeling demonstrated incorporation of 75Se into selenoproteins. UGA recoding was assessed individually for all expressed selenoproteins by RiboSeq. Analysis of ribosomes residing on UGA either at the A-site or the P-site revealed transcript-specific responses to SECISBP2 inactivation. We propose a new measure to assess UGA/Sec read-through in all selenoproteins, i.e. the proportion of ribosome protected fragments (RPF) carrying UGA adjacent to the P-site, pUGA. Omission of translation inhibitors like cycloheximide during preparation allowed to asses not only ribosomes with occupied A-sites, but also ribosomes with unoccupied A-sites, which are differentiated by footprint size. Considering UGA positions in the RPF and footprint size, new details of UGA/Sec re-definition in individual selenoproteins can be uncovered.

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.

SECISBP2 is a novel prognostic predictor that regulates selenoproteins in diffuse large B-cell lymphoma

AbstractThe overexpression of glutathione peroxidase 4 (GPX4; an enzyme that suppresses peroxidation of membrane phospholipids) is considered a poor prognostic predictor of diffuse large B-cell lymphoma (DLBCL). However, the mechanisms employed in GPX4 overexpression remain unknown. GPX4 is translated as a complete protein upon the binding of SECISBP2 to the selenocysteine insertion sequence (SECIS) on the 3′UTR of GPX4 mRNA. In this study, we investigated the expression of SECISBP2 and its subsequent regulation of GPX4 and TXNRD1 in DLBCL patients. Moreover, we determined the significance of the expression of these selenoproteins in vitro using MD901 and Raji cells. SECISBP2 was positive in 45.5% (75/165 cases) of DLBCL samples. The SECISBP2-positive group was associated with low overall survival (OS) as compared to the SECISBP2-negative group (P = 0.006). Similarly, the SECISBP2 and GPX4 or TXNRD1 double-positive groups (P < 0.001), as well as the SECISBP2, GPX4, and TXNRD1 triple-positive group correlated with poor OS (P = 0.001), suggesting that SECISBP2 may serve as an independent prognostic predictor for DLBCL (hazard ratio (HR): 2.693, P = 0.008). In addition, western blotting showed a decrease in GPX4 and TXNRD1 levels in SECISBP2-knockout (KO) MD901 and Raji cells. Oxidative stress increased the accumulation of reactive oxygen species in SECISBP2-KO cells (MD901; P < 0.001, Raji; P = 0.020), and reduced cell proliferation (MD901; P = 0.001, Raji; P = 0.030), suggesting that SECISBP2-KO suppressed resistance to oxidative stress. Doxorubicin treatment increased the rate of cell death in SECISBP2-KO cells (MD901; P < 0.001, Raji; P = 0.048). Removal of oxidative stress inhibited the altered cell death rate. Taken together, our results suggest that SECISBP2 may be a novel therapeutic target in DLBCL.SECISBP2 overexpression is an independent negative prognostic predictor in diffuse large B-cell lymphoma. Additionally, SECISBP2 positively correlates with selenoprotein expression. In vitro, SECISBP2 knockout increases intracellular reactive oxygen species accumulation via the downregulation of selenoproteins, inhibiting cell growth and promoting cell death after doxorubicin treatment. Therefore, SECISBP2 is a potential therapeutic target for malignant lymphoma.

Selecting cells expressing high levels of recombinant proteins using the GPI-anchored protein with selenocysteine system

Most biopharmaceutical proteins are produced in mammalian cells because they have the advantageous capacity for protein folding, assembly, and posttranslational modifications. To satisfy the increasing demand for these proteins for clinical purposes and studies, traditional methods to improve protein productivity have included gene amplification, host cell engineering, medium optimization, and screening methods. However, screening and selection of high-producing cell lines remain complex and time consuming. In this study, we established a glycosylphosphatidylinositol (GPI)-anchored protein with a selenocysteine (GPS) system to select cells producing high levels of target secretory proteins. Recombinant lysosomal acid lipase (LIPA) and α-galactosidase A (GALA) were fused with a GPI attachment signal sequence and a selenocysteine insertion sequence after an in-frame UGA codon. Under these conditions, most of the recombinant proteins were secreted into the culture medium, but some were found to be GPI-anchored proteins on the cell surface. When sodium selenite was supplied into the culture medium, the amount of GPI-anchored LIPA and GALA was increased. High-expressing cells were selected by detecting surface GPI-anchored LIPA. The GPI-anchored protein was then eliminated by knocking out the GPI biosynthesis gene PIGK, in these cells, all LIPA was in secreted form. Our system provides a promising method of isolating cells that highly express recombinant proteins from large cell populations.

Selenocysteine insertion sequence binding protein 2 (Sbp2) in the sex-specific regulation of selenoprotein gene expression in mouse pancreatic islets

Selenoproteins are a group of selenocysteine-containing proteins with major roles in cellular antioxidant defense and thyroid hormone metabolism. Selenoprotein expression is determined by hierarchical mechanisms that result in tissue-specific levels. Current data inadequately explain the abundance of various selenoproteins under normal and pathological conditions, including in pancreatic β-cells. Selenocysteine insertion sequence binding protein 2 (SBP2) is a critical protein in selenoprotein translation that also plays an essential role in stabilizing selenoprotein transcripts by antagonizing nonsense-mediated decay (NMD). Importantly, dysfunctional SBP2 is associated with endocrine disorders in humans. Here we describe the impact of induced Sbp2 deficiency in pancreatic β-cells on selenoprotein transcript profiles in the pancreatic islets of C57BL/6J mice. Sex differences were noted in control mice, in which female islets showed 5 selenoproteins decreased and one increased versus male islets. Induced Sbp2 deficiency in pancreatic β-cells altered expression of only 3 selenoprotein transcripts in male islets, whereas 14 transcripts were reduced in female islets. In all cases, decreased transcription was observed in genes known to be regulated by NMD. The differential impact of Sbp2 deletion on selenoprotein transcription between sexes suggests sex-specific hierarchical mechanisms of selenoprotein expression that may influence islet biology and consequentially metabolic disease risk.

The algal selenoproteomes

BackgroundSelenium is an essential trace element, and selenocysteine (Sec, U) is its predominant form in vivo. Proteins that contain Sec are selenoproteins, whose special structural features include not only the TGA codon encoding Sec but also the SECIS element in mRNA and the conservation of the Sec-flanking region. These unique features have led to the development of a series of bioinformatics methods to predict and research selenoprotein genes. There have been some studies and reports on the evolution and distribution of selenoprotein genes in prokaryotes and multicellular eukaryotes, but the systematic analysis of single-cell eukaryotes, especially algae, has been very limited.ResultsIn this study, we predicted selenoprotein genes in 137 species of algae by using a program we previously developed. More than 1000 selenoprotein genes were obtained. A database website was built to record these algae selenoprotein genes (www.selenoprotein.com). These genes belong to 42 selenoprotein families, including three novel selenoprotein gene families.ConclusionsThis study reveals the primordial state of the eukaryotic selenoproteome. It is an important clue to explore the significance of selenium for primordial eukaryotes and to determine the complete evolutionary spectrum of selenoproteins in all life forms.

The Interaction between Dietary Selenium Intake and Genetics in Determining Cancer Risk and Outcome.

There is considerable interest in the trace element selenium as a possible cancer chemopreventive dietary component, but supplementation trials have not indicated a clear benefit. Selenium is a critical component of selenium-containing proteins, or selenoproteins. Members of this protein family contain selenium in the form of selenocysteine. Selenocysteine is encoded by an in-frame UGA codon recognized as a selenocysteine codon by a regulatory element, the selenocysteine insertion sequence (SECIS), in the 3′-untranslated region of selenoprotein mRNAs. Epidemiological studies have implicated several selenoprotein genes in cancer risk or outcome based on associations between allelic variations and disease risk or mortality. These polymorphisms can be found in or near the SECIS or in the selenoprotein coding sequence. These variations both function to control protein synthesis and impact the efficiency of protein synthesis in response to the levels of available selenium. Thus, an individual’s genetic makeup and nutritional intake of selenium may interact to predispose them to acquiring cancer or affect cancer progression to lethality.

Tetrahymena Glutathione Peroxidase Family: A Comparative Analysis of These Antioxidant Enzymes and Differential Gene Expression to Metals and Oxidizing Agents.

In the present work, an extensive analysis of the putative glutathione peroxidases (GPx) of the eukaryotic microorganism model Tetrahymena thermophila is carried out. A comparative analysis with GPx present in other Tetrahymena species and other very taxonomically diverse ciliates is also performed. A majority of ciliate GPx have replaced the selenocysteine (Sec) by Cys in its catalytic center, so they can be considered as phospholipid hydroperoxide glutathione peroxidases (PHGPx). Selenocysteine insertion sequence (SECIS) elements have been detected in several ciliate GPx that do not incorporate Sec in their amino acid sequences, and conversely, in other ciliate GPx with Sec, no SECIS elements are detected. These anomalies are analyzed and discussed. From the phylogenetic analysis using the ciliate GPx amino acid sequences, the existence of extensive intra- and interspecific gene duplications that produced multiple GPx isoforms in each species is inferred. The ancestral character of the selenoproteins is also corroborated. The analysis by qRT-PCR of six selected T. thermophila GPx genes has shown a quantitative differential expression between them, depending on the stressor (oxidizing agents, apoptotic inducer or metals) and the time of exposure.

Characterization and expression analysis of seven selenoprotein genes in yellow catfish Pelteobagrus fulvidraco to dietary selenium levels

BackgroundSelenium (Se) appears in the selenoproteins in the form of selenocysteine (Sec) and is important for the growth and development of vertebrates. The present study characterized seven selenoproteins, consisting of the GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3 cDNAs in various tissues of yellow catfish, explored their regulation to dietary Se addition. Methods3′ and 5′ RACE PCR were used to clone full-length cDNA sequences of seven selenoprotein genes (GPX1, GPX3, GPX4, SELENOW, SELENOP, TXNRD2 and TXNRD3). Their molecular characterizations were analyzed, including conservative motifs and the SECIS elements. The phylogenetic trees were generated through neighbor-joining (NJ) method with MEGA 6.0 with 1000 bootstrap replications. Quantitative real-time PCR was used to explore their mRNA tissue distribution in the heart, anterior intestine, dorsal muscle, head kidney, gill, liver, brain, spleen and mesenteric fat. Yellow catfish (mixed sex) were fed diets with dietary Se contents at 0.03 (low Se), 0.25 (adequate Se) and 6.39 (high Se) mg Se/kg, respectively, for 12 weeks, and their spleen, kidney, testis and brain were used for the determination of the mRNA levels of the seven selenoproteins. ResultsThe seven selenoproteins had similar domains to their corresponding members of other vertebrates. They were widely expressed in nine tissues, including heart, liver, brain, spleen, head kidney, dorsal muscle, mesenteric fat, anterior intestine and gill, but showed tissue-dependent expression patterns. Dietary Se addition affected the expression of the seven genes in spleen, kidney, testis and brain tissues of yellow catfish. ConclusionTaken together, our study demonstrated the characterization, expression and regulation of seven selenoproteins, which increased our understanding of the biological functions of Se and selenoproteins in fish.

Control of Selenoprotein F Translation in Prostate Cancer

Abstract Objectives Selenoprotein F (SELENOF) is a selenocysteine-containing protein whose levels are responsive to selenium availability and are often lower in prostate cancer (PCa) compared to benign regions. Lowering SELENOF levels in cultured, non-transformed prostate cells results in these cells gaining a transformed phenotype, indicating that SELENOF may function as a tumor suppressor. The reasons for the loss of SELENOF in PCa remain unknown. SELENOF translation requires a selenocysteine insertion sequence (SECIS), a stem-loop structure within the 3’– untranslated region (UTR) of the SELENOF mRNA. The objective of this study is to determine if SECIS-mediated UGA recoding is impeded in PCa, resulting in lower SELENOF protein levels. Methods In order to establish a cell model for the decrease in SELENOF levels, SELENOF levels were determined by western blot analysis of protein from PC3 PCa cells and RWPE-1 immortalized but not tumorigenic prostate cells. SELENOF mRNA levels in each of these cell lines was subsequently determined by RT-qPCR. In order to determine the efficiency of UGA readthrough in PC3 and RWPE-1 cells, reporter constructs were generated in which an in-frame UGA was introduced into the open reading frame of firefly luciferase by in-vitro mutagenesis and the constructs included portions of the SELENOF 3’-UTR required for UGA recoding. Results SELENOF levels were 18 times lower in PC3 cells compared to the RWPE-1 cells, while mRNA levels were similar between the two. Using the generated reporter constructs, differences in UGA recoding efficiency in PC3 and RWPE-1 cells were apparent, as were unexpected contributions of portions of the 3’UTR sequences to UGA readthrough efficiency. Conclusions Differing levels of SELENOF seen in prostate tissues were recapitulated in a cell culture model, with there being significantly less protein in PC3 cells compared to RWPE-1 cells, without a comparable difference in mRNA. Changes in SELENOF levels due to altered regulation of UGA recoding may be a significant event in PCa progression. Funding Sources This work was supported by the University of Illinois at Chicago Liberal Arts and Sciences Undergraduate Research Initiative (LASURI) to SK and Department of Defense Prostate Cancer Research Program to AMD.

Why Multiples of 21? Why does Selenoprotein P Contain Multiple Selenocysteine Residues?

Background:In animals, the 21st amino acid selenocysteine is incorporated into a restricted subset of proteins by recoding of a UGA stop codon. This recoding requires a distinctive selenocysteine insertion sequence in selenoprotein encoding mRNAs, trans-acting factors and in most cases, adequate dietary intake of selenium. With one exception, selenoproteins contain a single selenocysteine, which is incorporated with low translational efficiency. The exception is selenoprotein P, which in some species is predicted to contain as many as 132 selenocysteines and which is considered to play roles in selenium transport and storage.Objective:This study aimed to develop comparative physiological and evolutionary perspectives on the function(s) of selenoprotein P.Method:The review of the literature on the roles of selenoprotein P in diverse animals.Results:Selenoprotein P contains multiple selenocysteines, making it energetically costly to produce. Furthermore, it is often associated with detrimental effects to the animals that produce it. Possible benefits that outweigh these costs include the general storage and transport of selenium; the transport of both toxic and useful metal ions; and specific functions in reproduction and in the nervous system.Conclusion:A probable reconciliation of the negative effects of producing Selenoprotein P is its benefit in terms of promoting reproductive success.

Generation of Recombinant Mammalian Selenoproteins through Genetic Code Expansion with Photocaged Selenocysteine.

Selenoproteins contain the amino acid selenocysteine (Sec) and are found in all domains of life. The functions of many selenoproteins are poorly understood, partly due to difficulties in producing recombinant selenoproteins for cell-biological evaluation. Endogenous mammalian selenoproteins are produced through a noncanonical translation mechanism requiring suppression of the UGA stop codon and a Sec insertion sequence (SECIS) element in the 3' untranslated region of the mRNA. Here, recombinant selenoproteins are generated in mammalian cells through genetic code expansion, circumventing the requirement for the SECIS element and selenium availability. An engineered orthogonal E. coli leucyl-tRNA synthetase/tRNA pair is used to incorporate a photocaged Sec (DMNB-Sec) at the UAG amber stop codon. DMNB-Sec is successfully incorporated into GFP and uncaged by irradiation of living cells. Furthermore, DMNB-Sec is used to generate the native selenoprotein methionine-R-sulfoxide reductase B1 (MsrB1). Importantly, MsrB1 is shown to be catalytically active after uncaging, constituting the first use of genetic code expansion to generate a functional selenoprotein in mammalian systems. The ability to site-specifically introduce Sec directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

Loss of Selenoprotein F in Prostate Cancer

ObjectiveSelenium‐containing proteins, or selenoproteins, are likely to play a role in prostate cancer progression and mortality. Selenoprotein F (SELENOF) is a selenium‐containing protein implicated in prostate cancer etiology due to its dramatic reduction in prostate cancer tissue when compared to adjacent benign regions. The reduction in SELENOF levels in immortalized prostate epithelial cells results in phenotypic features associated with cellular transformation, indicating that SELENOF functions as a tumor suppressor in the prostate. The mechanism behind the reduction of SELENOF levels in prostate cancer remains unknown, although it is suspected that it involves events at the level of protein synthesis, based on work done in tissue culture cells.SELENOF synthesis is controlled by sequences within the 3′ untranslated sequences of SELENOF mRNA known as selenocysteine insertion sequences (SECIS) that are required for the recognition of in‐frame UGA codons as the selenocysteine amino acid. Elongation initiation factor 4a3 (eIF4a3) binds SECIS during times of low selenium availability and suppresses the synthesis of several selenoproteins. Moreover, higher levels of eIF4a3 are associated with increased grade of prostate cancer. The objective of this study is to determine if elevated eIF4a3 levels suppress SELENOF synthesis and contribute to prostate cancer progression.MethodologyIn order to establish a model for SELENOF loss in prostate‐derived cells, SELENOF levels were determined by western blot and mRNA levels by RT‐PCR in PC3 prostate cancer cells and non‐tumorigenic RWPE‐1 cells. Reporter constructs in which the production of light‐emitting luciferase requires SECIS function to recognize a UGA placed in the open reading frame of the luciferase gene were generated by in vitro mutagenesis. An eIF4a3 overexpression construct was used to overexpress that protein in transfected cells to directly determine its impact on SELENOF synthesis.ResultsSELENOF levels were 18‐fold lower in PC3 cells when compared to RWPE‐1 cells, although mRNA levels were similar between the two cell lines. Using the luciferase reporter constructs described above, it was determined that the readthrough of the UGA in the luciferase gene and supported by the SELENOF SECIS element was more efficient in the RWPE‐1 cells as compared to PC3 cells. In addition, over‐expression of eIF4a3 in RWPE‐1 cells was achieved and the consequences on SELENOF translation and the transformed phenotype are currently under investigation.ConclusionThe loss of SELENOF in prostate cancer is likely to occur at the level of translation and mediated through molecular events involving the SECIS element. A likely candidate for how this occurs has been identified as eIF4a3, an RNA helicase whose levels are elevated in prostate cancer. Enhancing SELENOF levels and/or diminishing eIF4a3 are potentially novel approaches to treat prostate cancers that are no longer responsive to existing treatments.Support or Funding InformationThis work was supported by grant PC170236 from the Department of Defense Prostate Cancer Research Program

Cul5-type Ubiquitin Ligase KLHDC1 Contributes to the Elimination of Truncated SELENOS Produced by Failed UGA/Sec Decoding.

SummaryThe UGA codon signals protein translation termination, but it can also be translated into selenocysteine (Sec, U) to produce selenocysteine-containing proteins (selenoproteins) by dedicated machinery. As Sec incorporation can fail, Sec-containing longer and Sec-lacking shorter proteins co-exist. Cul2-type ubiquitin ligases were recently shown to destabilize such truncated proteins; however, which ubiquitin ligase targets truncated proteins for degradation remained unclear. We report that the Cul5-type ubiquitin ligase KLHDC1 targets truncated SELENOS, a selenoprotein, for proteasomal degradation. SELENOS is involved in endoplasmic reticulum (ER)-associated degradation, which is linked to reactive oxygen species (ROS) production, and the knockdown of KLHDC1 in U2OS cells decreased ER stress-induced cell death. Knockdown of SELENOS increased the cell population with lower ROS levels. Our findings reveal that, in addition to Cul2-type ubiquitin ligases, KLHDC1 is involved in the elimination of truncated oxidoreductase-inactive SELENOS, which would be crucial for maintaining ROS levels and preventing cancer development.

Chemical Biology Approaches to Interrogate the Selenoproteome.

Selenoproteins are the family of proteins that contain the amino acid selenocysteine. Many selenoproteins, including glutathione peroxidases and thioredoxin reductases, play a role in maintaining cellular redox homeostasis. There are a number of examples of homologues of selenoproteins that utilize cysteine residues, raising the question of why selenocysteines are utilized. One hypothesis is that incorporation of selenocysteine protects against irreversible overoxidation, typical of cysteine-containing homologues under high oxidative stress. Studies of selenocysteine function are hampered by challenges both in detection and in recombinant expression of selenoproteins. In fact, about half of the 25 known human selenoproteins remain uncharacterized. Historically, selenoproteins were first detected via labeling with radioactive 75Se or by use of inductively coupled plasma-mass spectrometry to monitor nonradioactive selenium. More recently, tandem mass-spectrometry techniques have been developed to detect selenocysteine-containing peptides. For example, the isotopic distribution of selenium has been used as a unique signature to identify selenium-containing peptides from unenriched proteome samples. Additionally, selenocysteine-containing proteins and peptides were selectively enriched using thiol-reactive electrophiles by exploiting the increased reactivity of selenols relative to thiols, especially under low pH conditions. Importantly, the reactivity-based enrichment of selenoproteins can differentiate between oxidized and reduced selenoproteins, providing insight into the activity state. These mass spectrometry-based selenoprotein detection approaches have enabled (1) production of selenoproteome expression atlases, (2) identification of aging-associated changes in selenoprotein expression, (3) characterization of selenocysteine reactivity across the selenoprotein family, and (4) interrogation of selenoprotein targets of small-molecule drugs. Further investigations of selenoprotein function would benefit from recombinant expression of selenoproteins. However, the endogenous mechanism of selenoprotein production makes recombinant expression challenging. Primarily, selenocysteine is biosynthesized on its own tRNA, is dependent on multiple enzymatic steps, and is highly sensitive to selenium concentrations. Furthermore, selenocysteine is encoded by the stop codon UGA, and suppression of that stop codon requires a selenocysteine insertion sequence element in the selenoprotein mRNA. In order to circumvent the low efficiency of the endogenous machinery, selenoproteins have been produced in vitro through native chemical ligation and expressed protein ligation. Attempts have also been made to engineer the endogenous machinery for increased efficiency, including recoding the selenocysteine codon, and engineering the tRNA and the selenocysteine insertion sequence element. Alternatively, genetic code expansion can be used to generate selenoproteins. This approach allows for selenoprotein production directly within its native cellular environment, while bypassing the endogenous selenocysteine incorporation machinery. Furthermore, by incorporating a caged selenocysteine by genetic code expansion, selenoprotein activity can be spatially and temporally controlled. Genetic code expansion has allowed for the expression and uncaging of human selenoproteins in E. coli and more recently in mammalian cells. Together, advances in selenoprotein detection and expression should enable a better understanding of selenoprotein function and provide insight into the necessity for selenocysteine production.

Ribosome profiling of selenoproteins in vivo reveals consequences of pathogenic Secisbp2 missense mutations

Recoding of UGA codons as selenocysteine (Sec) codons in selenoproteins depends on a selenocysteine insertion sequence (SECIS) in the 3'-UTR of mRNAs of eukaryotic selenoproteins. SECIS-binding protein 2 (SECISBP2) increases the efficiency of this process. Pathogenic mutations in SECISBP2 reduce selenoprotein expression and lead to phenotypes associated with the reduction of deiodinase activities and selenoprotein N expression in humans. Two functions have been ascribed to SECISBP2: binding of SECIS elements in selenoprotein mRNAs and facilitation of co-translational Sec insertion. To separately probe both functions, we established here two mouse models carrying two pathogenic missense mutations in Secisbp2 previously identified in patients. We found that the C696R substitution in the RNA-binding domain abrogates SECIS binding and does not support selenoprotein translation above the level of a complete Secisbp2 null mutation. The R543Q missense substitution located in the selenocysteine insertion domain resulted in residual activity and caused reduced selenoprotein translation, as demonstrated by ribosomal profiling to determine the impact on UGA recoding in individual selenoproteins. We found, however, that the R543Q variant is thermally unstable in vitro and completely degraded in the mouse liver in vivo, while being partially functional in the brain. The moderate impairment of selenoprotein expression in neurons led to astrogliosis and transcriptional induction of genes associated with immune responses. We conclude that differential SECISBP2 protein stability in individual cell types may dictate clinical phenotypes to a much greater extent than molecular interactions involving a mutated amino acid in SECISBP2.