In silico identification of genes involved in selenium metabolism: evidence for a third selenium utilization trait

<p>Abstract</p> <p>Background</p> <p>Selenium (Se) is a trace element that occurs in proteins in the form of selenocysteine (Sec) and in tRNAs in the form of selenouridine (SeU). Selenophosphate synthetase (SelD) is required for both utilization traits. However, previous research also revealed SelDs in two organisms lacking Sec and SeU, suggesting a possible additional use of Se that is dependent on SelD.</p> <p>Results</p> <p>In this study, we conducted comparative genomics and phylogenetic analyses to characterize genes involved in Se utilization. Candidate genes identified included SelA/SelB and YbbB that define Sec and SeU pathways, respectively, and NADH oxidoreductase that is predicted to generate a SelD substrate. In addition, among 227 organisms containing SelD, 10 prokaryotes were identified that lacked SelA/SelB and YbbB. Investigation of <it>selD </it>neighboring genes in these organisms revealed a SirA-like protein and two hypothetical proteins HP1 and HP2 that were strongly linked to a novel Se utilization. With these new signature proteins, 32 bacteria and archaea were found that utilized these proteins, likely as part of the new Se utilization trait. Metabolic labeling of one organism containing an orphan SelD, <it>Enterococcus faecalis</it>, with ⁷⁵Se revealed a protein containing labile Se species that could be released by treatment with reducing agents, suggesting non-Sec utilization of Se in this organism.</p> <p>Conclusion</p> <p>These studies suggest the occurrence of a third Se utilization trait in bacteria and archaea.</p

UGA codon position-dependent incorporation of selenocysteine into mammalian selenoproteins

It is thought that the SelenoCysteine Insertion Sequence (SECIS) element and UGA codon are sufficient for selenocysteine (Sec) insertion. However, we found that UGA supported Sec insertion only at its natural position or in its close proximity in mammalian thioredoxin reductase 1 (TR1). In contrast, Sec could be inserted at any tested position in mammalian TR3. Replacement of the 3′-UTR of TR3 with the corresponding segment of a Euplotes crassus TR restricted Sec insertion into the C-terminal region, whereas the 3′-UTR of TR3 conferred unrestricted Sec insertion into E. crassus TR, in which Sec insertion is normally limited to the C-terminal region. Exchanges of 3′-UTRs between mammalian TR1 and E. crassus TR had no effect, as both proteins restricted Sec insertion. We further found that these effects could be explained by the use of selenoprotein-specific SECIS elements. Examination of Sec insertion into other selenoproteins was consistent with this model. The data indicate that mammals evolved the ability to limit Sec insertion into natural positions within selenoproteins, but do so in a selenoprotein-specific manner, and that this process is controlled by the SECIS element in the 3′-UTR

Adjustments, extinction, and remains of selenocysteine incorporation machinery in the nematode lineage

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date. After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International).Selenocysteine (Sec) is encoded by an UGA codon with the help of a SECIS element present in selenoprotein mRNAs. SECIS-binding protein (SBP2/SCBP-2) mediates Sec insertion, but the roles of its domains and the impact of its deficiency on Sec insertion are not fully understood. We used Caenorhabditis elegans to examine SBP2 function since it possesses a single selenoprotein, thioredoxin reductase-1 (TRXR-1). All SBP2 described so far have an RNA-binding domain (RBD) and a Secincorporation domain (SID). Surprisingly, C. elegans SBP2 lacks SID and consists only of an RBD. An sbp2 deletion mutant strain ablated Sec incorporation demonstrating SBP2 essentiality for Sec incorporation. Further in silico analyses of nematode genomes revealed conservation of SBP2 lacking SID and maintenance of Sec incorporation linked to TRXR-1. Remarkably, parasitic plant nematodes lost the ability to incorporate Sec, but retained SecP43, a gene associated with Sec incorporation. Interestingly, both selenophosphate synthetase (SPS) genes are absent in plant parasitic nematodes, while only Cys-containing SPS2 is present in Sec-incorporating nematodes. Our results indicate that C. elegans and the nematode lineage provide key insights into Sec incorporation and the evolution of Sec utilization trait, selenoproteomes, selenoproteins, and Sec residues. Finally, our study provides evidence of noncanonical translation initiation in C. elegans, not previously known for this well-established animal model.This work was supported by Universidad de la República, Uruguay (Grant Number 418 to G.S., PhD fellowship to L.O.); Asociación Española de Cooperación Internacional (C/7646/07 to A.M.-V. and G.S.; A/016083/08 to A.M.-V. and G.S.); Asociación Universitaria Iberoamericana de Posgrado and Agencia Nacional de Innovación e Investigación (BE_POS_2009_183 and BE_POS_2010_2160 to L.O.), and was partially funded by FOCEM (MERCOSUR Structural Convergence Fund), [COF 03/11].Peer Reviewe

Regulation of Selenocysteine Content of Human Selenoprotein P by Dietary Selenium and Insertion of Cysteine in Place of Selenocysteine

Selenoproteins are a unique group of proteins that contain selenium in the form of selenocysteine (Sec) co-translationally inserted in response to a UGA codon with the help of cis- and trans-acting factors. Mammalian selenoproteins contain single Sec residues, with the exception of selenoprotein P (SelP) that has 7–15 Sec residues depending on species. Assessing an individual’s selenium status is important under various pathological conditions, which requires a reliable selenium biomarker. Due to a key role in organismal selenium homeostasis, high Sec content, regulation by dietary selenium, and availability of robust assays in human plasma, SelP has emerged as a major biomarker of selenium status. Here, we found that Cys is present in various Sec positions in human SelP. Treatment of cells expressing SelP with thiophosphate, an analog of the selenium donor for Sec synthesis, led to a nearly complete replacement of Sec with Cys, whereas supplementation of cells with selenium supported Sec insertion. SelP isolated directly from human plasma had up to 8% Cys inserted in place of Sec, depending on the Sec position. These findings suggest that a change in selenium status may be reflected in both SelP concentration and its Sec content, and that availability of the SelP-derived selenium for selenoprotein synthesis may be overestimated under conditions of low selenium status due to replacement of Sec with Cys

2\u27-O-Methyl at 20-mer Guide Strand 3\u27 Termini May Negatively Affect Target Silencing Activity of Fully Chemically Modified siRNA

Small interfering RNAs (siRNAs) have the potential to treat a broad range of diseases. siRNAs need to be extensively chemically modified to improve their bioavailability, safety, and stability in vivo. However, chemical modifications variably impact target silencing for different siRNA sequences, making the activity of chemically modified siRNA difficult to predict. Here, we systematically evaluated the impact of 3\u27 terminal modifications (2\u27-O-methyl versus 2\u27-fluoro) on guide strands of different length and showed that 3\u27 terminal 2\u27-O-methyl modification negatively impacts activity for \u3e60% of siRNA sequences tested but only in the context of 20- and not 19- or 21-nt-long guide strands. These results indicate that sequence, modification pattern, and structure may cooperatively affect target silencing. Interestingly, the introduction of an extra 2\u27-fluoro modification in the seed region at guide strand position 5, but not 7, may partially compensate for the negative impact of 3\u27 terminal 2\u27-O-methyl modification. Molecular modeling analysis suggests that 2\u27-O-methyl modification may impair guide strand interactions within the PAZ domain of argonaute-2, which may affect target recognition and cleavage, specifically when guide strands are 20-nt long. Our findings emphasize the complex nature of modified RNA-protein interactions and contribute to design principles for chemically modified siRNAs

5-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo

5-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5-vinylphosphonate confers novel properties to siRNAs. Specifically, 5-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5-to-3 exonucleases. Delivery of conjugated siRNAs requires extensive chemical modifications to achieve stability in vivo. Because chemically modified siRNAs are poor substrates for phosphorylation by kinases, and 5-phosphate is required for loading into RNA-induced silencing complex, the synthetic addition of a 5-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5-phosphate is removed within 2 hours. The 5-phosphate mimic 5-(E)-vinylphosphonate stabilizes the 5 end of the guide strand by protecting it from phosphatases and 5-to-3 exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5-(E)-vinylphosphonate stabilizes 5 phosphate, thereby enabling systemic delivery to and silencing in kidney and heart

Analyses of Fruit Flies That Do Not Express Selenoproteins or Express the Mouse Selenoprotein, Methionine Sulfoxide Reductase B1, Reveal a Role of Selenoproteins in Stress Resistance

Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with 75Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms

Reduced Utilization of Selenium by Naked Mole Rats Due to a Specific Defect in GPx1 Expression

Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (\u3e28 years). It is also not known to develop cancer. In the current work, tissue imaging by x-ray fluorescence microscopy and direct analyses of trace elements revealed low levels of selenium in the MR liver and kidney, whereas MR and mouse brains had similar selenium levels. This effect was not explained by uniform selenium deficiency because methionine sulfoxide reductase activities were similar in mice and MR. However, glutathione peroxidase activity was an order of magnitude lower inMRliver and kidney than in mouse tissues. In addition, metabolic labeling of MR cells with 75Se revealed a loss of the abundant glutathione peroxidase 1 (GPx1) band, whereas other selenoproteins were preserved. To characterize theMRselenoproteome, we sequenced its liver transcriptome. Gene reconstruction revealed standard selenoprotein sequences except for GPx1, which had an early stop codon, and SelP, which had low selenocysteine content. When expressed inHEK293cells,MRGPx1waspresent in low levels,and its expression could be rescued neither by removing the early stop codon nor by replacing its SECIS element. In addition, GPx1 mRNAwas present in lower levels inMRliver than in mouse liver. To determine if GPx1 deficiency could account for the reduced selenium content, we analyzed GPx1 knock-out mice and found reduced selenium levels in their livers and kidneys. Thus, MR is characterized by the reduced utilization of selenium due to a specific defect in GPx1 expression

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

Abstract Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery

Thioredoxin reductase in redox regulation and genetic code

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains intracellular redox balance. Both cytosolic (TR1, Trx1) and mitochondrial (TR3, Trx2) enzymes are essential in mammals. We identified and characterized in detail the subcellular localization of three TR3 forms that are generated by alternative first exon splicing and differ in their N-terminal sequences. Recombinant selenoprotein forms of TR1 and TR3 were generated and regulation of their activities by metal ions investigated. Both enzymes were inhibited by zinc, but not by calcium or cobalt. In addition, we developed a proteomic method to identify targets of mammalian TRs. Trx1 was the major target for both TR1 and TR3 in rat and mouse liver cytosolic fractions and could be specifically enriched from tissue extracts and body liquids on TR affinity columns. To examine the roles of TRs in redox regulation, knockout mice in which selenocysteine (Sec) tRNA gene was disrupted specifically in the liver were employed. We found that the levels of TR1 and TR3 were reduced in the knockout samples, whereas the levels of Trx1 and Trx2 were elevated; however, Trxs were in the oxidized state in the knockout samples. Overall, these data provided evidence for key roles of TRs in redox homeostasis. In ciliated protozoa Euplotes crassus, cysteine (Cys) is encoded by three codons, UGA, UGU and UGC. UGA is a stop signal in the universal genetic code, and this codon can also code for the 21st amino acid, Sec, in all three domains of life. We found that in Euplotes crassus the UGA codon specifies the insertion of both Cys and Sec. We characterized the E. crassus thioredoxin reductase gene, in which the first six UGA codons code for Cys and the seventh for Sec. Our data show that E. crassus utilizes UGA for insertion of both Cys and Sec, establishing it as the first known organism that utilizes one codon to code unambiguously for two amino acids