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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′ terminal modifications (2′-O-methyl versus 2′-fluoro) on guide strands of different length and showed that 3′ terminal 2′-O-methyl modification negatively impacts activity for >60% 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′-fluoro modification in the seed region at guide strand position 5, but not 7, may partially compensate for the negative impact of 3′ terminal 2′-O-methyl modification. Molecular modeling analysis suggests that 2′-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., Graphical Abstract, Davis et al. demonstrate reduced target silencing activity for fully chemically modified siRNAs with 3′ guide strand terminal 2′-O-methyl versus 2′-fluoro modification. They show that this negative impact is specific to siRNAs with 20-mer guide strands and can be partially compensated by 2′-fluoro modification at guide strand position 5.

Thioredoxin/glutathione reductase (TGR, TXNRD3) is a thiol oxidoreductase of unknown function composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent enzyme evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

Thioredoxin/glutathione reductase (TXNRD3) is a selenoprotein composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent thiol oxidoreductase evolved through gene duplication within the Txnrd family, is expressed in the testes, and can reduce both thioredoxin and glutathione in vitro; however, the function of this enzyme remains unknown. To characterize the function of TXNRD3 in vivo, we generated a strain of mice bearing deletion of Txnrd3 gene. We show that these Txnrd3 knockout mice are viable and without discernable gross phenotypes, and also that TXNRD3 deficiency leads to fertility impairment in male mice. We found that Txnrd3 knockout animals exhibited a lower fertilization rate in vitro, a sperm movement phenotype, and an altered thiol redox status in sperm cells. Proteomic analyses further revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of sperm quality control. Taken together, these results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington’s disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders. A divalent siRNA architecture enables sustained silencing of gene expression in deep regions of the brain.

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.

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.

The ribosome can change its reading frame during translation in a process known as programmed ribosomal frameshifting. These rare events are supported by complex mRNA signals. However, we found that the ciliates Euplotes crassus and Euplotes focardii exhibit widespread frameshifting at stop codons. 47 different codons preceding stop signals resulted in either +1 or +2 frameshifts, and +1 frameshifting at AAA was the most frequent. The frameshifts showed unusual plasticity and rapid evolution, and had little influence on translation rates. The proximity of a stop codon to the 3' mRNA end, rather than its occurrence or sequence context, appeared to designate termination. Thus, a 'stop codon' is not a sufficient signal for translation termination, and the default function of stop codons in Euplotes is frameshifting, whereas termination is specific to certain mRNA positions and probably requires additional factors.

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene(®) branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo.

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1 mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia.

The trace element selenium (Se) is incorporated into proteins as the amino acid selenocysteine (Sec), which is cotranslationally inserted into specific proteins in response to a UGA codon. Proteins containing Sec at these specific positions are called selenoproteins. Most selenoproteins function as oxidoreductases, while some serve other important functions. There are 25 known selenoprotein genes in humans and 24 in mice. The use of Sec allows selenoproteins to be detected by a convenient method involving metabolic labeling with

The trace element selenium (Se) is incorporated into proteins as the amino acid selenocysteine (Sec), which is cotranslationally inserted into specific proteins in response to a UGA codon. Proteins containing Sec at these specific positions are called selenoproteins. Most selenoproteins function as oxidoreductases, while some serve other important functions. There are 25 known selenoprotein genes in humans and 24 in mice. The use of Sec allows selenoproteins to be detected by a convenient method involving metabolic labeling with 75Se. Labeling of cells and whole animals are used for the examination of selenoprotein expression profiles and the investigation of selenoprotein functions. In mammals, nonspecific 75Se insertion is very low, and sensitivity and specificity of selenoprotein detection approaches that of Western blotting. This method allows for the examination of selenoprotein expression and Se metabolism in model and non-model organisms. Herein, we describe experimental protocols for analyzing selenoproteins by metabolic labeling with 75Se both in vitro and in vivo. As an example, the procedure for metabolic labeling of HEK293T human embryonic kidney cells is described in detail. This approach remains a method of choice for the detection of selenoproteins in diverse settings.

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 0 -UTR of TR3 with the corresponding segment of a Euplotes crassus TR restricted Sec insertion into the C-terminal region, whereas the 3 0 -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 0 -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 selenoproteinspecific manner, and that this process is controlled by the SECIS element in the 3 0 -UTR.

Antibiotics target bacteria by interfering with essential processes such as translation, but their effects on translation in mammalian cells are less well characterized. We found that doxycycline, chloramphenicol, and Geneticin (G418) interfered with insertion of selenocysteine (Sec), which is encoded by the stop codon, UGA, into selenoproteins in murine EMT6 cells. Treatment of EMT6 cells with these antibiotics reduced enzymatic activities and Sec insertion into thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1). However, these proteins were differentially affected due to varying errors in Sec insertion at UGA. In the presence of doxycycline, chloramphenicol, or G418, the Sec-containing form of TR1 decreased, whereas the arginine-containing and truncated forms of this protein increased. We also detected antibiotic-specific misinsertion of cysteine and tryptophan. Furthermore, misinsertion of arginine in place of Sec was commonly observed in GPx1 and glutathione peroxidase 4. TR1 was the most affected and GPx1 was the least affected by these translation errors. These observations were consistent with the differential use of two Sec tRNA isoforms and their distinct roles in supporting accuracy of Sec insertion into selenoproteins. The data reveal widespread errors in inserting Sec into proteins and in dysregulation of selenoprotein expression and function upon antibiotic treatment.

Selenium (Se) has long been known for its cancer prevention properties, but the molecular basis remains unclear. The principal questions in assessing the effect of dietary Se in cancer are whether selenoproteins, small molecule selenocompounds, or both, are involved, and under which conditions and genotypes Se may be protective. In this study, we examined diethylnitrosamine-induced hepatocarcinogenesis in mice lacking a subset of selenoproteins due to expression of a mutant selenocysteine tRNA gene (Trsp (A37G) mice). To uncouple the effects of selenocompounds and selenoproteins, these animals were examined at several levels of dietary Se. Our analysis revealed that tumorigenesis in Trsp (A37G) mice maintained on the adequate Se diet was increased. However, in the control, wild-type mice, both Se deficiency and high Se levels protected against tumorigenesis. We further found that the Se-deficient diet induced severe neurological phenotypes in Trsp A37G mice. Surprisingly, a similar phenotype could be induced in these mice at high dietary Se intake. Overall, our results show a complex role of Se in chemically induced hepatocarcinogenesis, which involves interaction among selenoproteins, selenocompounds and toxins, and depends on genotype and background of the animals.

Naked mole rat (MR) Heterocephalus glaber is a rodent model of delayed aging because of its unusually long life span (28 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 in MR liver and kidney than in mouse tissues. In addition, metabolic labeling of MR cells with (75)Se revealed a loss of the abundant glutathione peroxidase 1 (GPx1) band, whereas other selenoproteins were preserved. To characterize the MR selenoproteome, 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 in HEK 293 cells, MR GPx1 was present 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 mRNA was present in lower levels in MR liver 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.

The biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec tRNA, designated tRNA([Ser]Sec), is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase to form O-phosphoseryl-tRNA([Ser]Sec). Sec synthase (SecS) then uses O-phosphoseryl-tRNA([Ser]Sec) and the active donor of selenium, selenophosphate, to form Sec-tRNA([Ser]Sec). Selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase 2 (SPS2). Sec was the last protein amino acid in eukaryotes whose biosynthesis had not been established and the only known amino acid in eukaryotes whose biosynthesis occurs on its tRNA. Interestingly, sulfide can replace selenide to form thiophosphate in the SPS2-catalyzed reaction that can then react with O-phosphoseryl-tRNA([Ser]Sec) in the presence of SecS to form cysteine-(Cys-)tRNA([Ser]Sec). This novel pathway of Cys biosynthesis results in Cys being decoded by UGA and replacing Sec in normally selenium-containing proteins (selenoproteins). The selenoprotein, thioredoxin reductase 1 (TR1), was isolated from cells in culture and from mouse liver for analysis of Cys/Sec replacement by MS. The level of Cys/Sec replacement in TR1 was proportional to the level of selenium in the diet of the mice. Elucidation of the biosynthesis of Sec and Sec/Cys replacement provides novel ways of regulating selenoprotein functions and ultimately better understanding of the biological roles of dietary selenium.

Cysteine (Cys) is inserted into proteins in response to UGC and UGU codons. Herein, we show that supplementation of mammalian cells with thiophosphate led to targeted insertion of Cys at the UGA codon of thioredoxin reductase 1 (TR1). This Cys was synthesized by selenocysteine (Sec) synthase on tRNA [Ser]Sec and its insertion was dependent on the Sec insertion sequence element in the 3′UTR of TR1 mRNA. The substrate for this reaction, thiophosphate, was synthesized by selenophosphate synthetase 2 from ATP and sulfide and reacted with phosphoseryl-tRNA [Ser]Sec to generate Cys-tRNA [Ser]Sec . Cys was inserted in vivo at UGA codons in natural mammalian TRs, and this process was regulated by dietary selenium and availability of thiophosphate. Cys occurred at 10% of the Sec levels in liver TR1 of mice maintained on a diet with normal amounts of selenium and at 50% in liver TR1 of mice maintained on a selenium deficient diet. These data reveal a novel Sec machinery-based mechanism for biosynthesis and insertion of Cys into protein at UGA codons and suggest new biological functions for thiophosphate and sulfide in mammals.

Platyhelminth parasites are a major health problem in developing countries. In contrast to their mammalian hosts, platyhelminth thiol-disulfide redox homeostasis relies on linked thioredoxin-glutathione systems, which are fully dependent on thioredoxin-glutathione reductase (TGR), a promising drug target. TGR is a homodimeric enzyme comprising a glutaredoxin domain and thioredoxin reductase (TR) domains with a C-terminal redox center containing selenocysteine (Sec). In this study, we demonstrate the existence of functional linked thioredoxin-glutathione systems in the cytosolic and mitochondrial compartments of Echinococcus granulosus, the platyhelminth responsible for hydatid disease. The glutathione reductase (GR) activity of TGR exhibited hysteretic behavior regulated by the [GSSG]/[GSH] ratio. This behavior was associated with glutathionylation by GSSG and abolished by deglutathionylation. The Km and kcat values for mitochondrial and cytosolic thioredoxins (9.5 μm and 131 s–1, 34 μm and 197 s–1, respectively) were higher than those reported for mammalian TRs. Analysis of TGR mutants revealed that the glutaredoxin domain is required for the GR activity but did not affect the TR activity. In contrast, both GR and TR activities were dependent on the Sec-containing redox center. The activity loss caused by the Sec-to-Cys mutation could be partially compensated by a Cys-to-Sec mutation of the neighboring residue, indicating that Sec can support catalysis at this alternative position. Consistent with the essential role of TGR in redox control, 2.5 μm auranofin, a known TGR inhibitor, killed larval worms in vitro. These studies establish the selenium- and glutathione-dependent regulation of cytosolic and mitochondrial redox homeostasis through a single TGR enzyme in platyhelminths.

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.

SelS (Selenoprotein S) is a selenocysteine-containing protein with roles in ER (endoplasmic reticulum) function and inflammation. It has been implicated in ERAD (ER-associated protein degradation), and clinical studies revealed an association of its promoter polymorphism with cytokine levels and human diseases. However, the pathways and interacting proteins that could shed light on pathogenesis of SelS-associated diseases have not been studied systematically. We performed a large-scale affinity isolation of human SelS and its mutant forms and analysed the proteins that interact with them. All previously known SelS targets and nearly two hundred additional proteins were identified that were remarkably enriched for various multiprotein complexes. Subsequent chemical cross-linking experiments identified the specific interacting sites in SelS and its several targets. Most of these interactions involved coiled-coil domains. The data suggest that SelS participates in intracellular membrane transport and maintenance of protein complexes by anchoring them to the ER membrane.

Mammals differ more than 100-fold in maximum lifespan, which can be altered in either direction during evolution, but the molecular basis for natural changes in longevity is not understood. Divergent evolution of mammals also led to extensive changes in gene expression within and between lineages. To understand the relationship between lifespan and variation in gene expression, we carried out RNA-seq-based gene expression analyses of liver, kidney, and brain of 33 diverse species of mammals. Our analysis uncovered parallel evolution of gene expression and lifespan, as well as the associated life-history traits, and identified the processes and pathways involved. These findings provide direct insights into how nature reversibly adjusts lifespan and other traits during adaptive radiation of lineages.

S-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA(Sec) to the Um34 form. The formation of methylated tRNA(Sec) facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA(Sec) and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA(Sec) hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.

Strict one-to-one correspondence between codons and amino acids is thought to be an essential feature of the genetic code. However, we report that one codon can code for two different amino acids with the choice of the inserted amino acid determined by a specific 3′ untranslated region structure and location of the dual-function codon within the messenger RNA (mRNA). We found that the codon UGA specifies insertion of selenocysteine and cysteine in the ciliate Euplotes crassus , that the dual use of this codon can occur even within the same gene, and that the structural arrangements of Euplotes mRNA preserve location-dependent dual function of UGA when expressed in mammalian cells. Thus, the genetic code supports the use of one codon to code for multiple amino acids.

The naked mole rat (Heterocephalus glaber) is a strictly subterranean, extraordinarily long-lived eusocial mammal. Although it is the size of a mouse, its maximum lifespan exceeds 30 years, making this animal the longest-living rodent. Naked mole rats show negligible senescence, no age-related increase in mortality, and high fecundity until death. In addition to delayed ageing, they are resistant to both spontaneous cancer and experimentally induced tumorigenesis. Naked mole rats pose a challenge to the theories that link ageing, cancer and redox homeostasis. Although characterized by significant oxidative stress, the naked mole rat proteome does not show age-related susceptibility to oxidative damage or increased ubiquitination. Naked mole rats naturally reside in large colonies with a single breeding female, the 'queen', who suppresses the sexual maturity of her subordinates. They also live in full darkness, at low oxygen and high carbon dioxide concentrations, and are unable to sustain thermogenesis nor feel certain types of pain. Here we report the sequencing and analysis of the naked mole rat genome, which reveals unique genome features and molecular adaptations consistent with cancer resistance, poikilothermy, hairlessness and insensitivity to low oxygen, and altered visual function, circadian rythms and taste sensing. This information provides insights into the naked mole rat's exceptional longevity and ability to live in hostile conditions, in the dark and at low oxygen. The extreme traits of the naked mole rat, together with the reported genome and transcriptome information, offer opportunities for understanding ageing and advancing other areas of biological and biomedical research.

The biosynthetic pathway of selenocysteine (Sec), the 21st amino acid in the genetic code, has been established in eukaryotes and archaea using comparative genomic and experimental approaches. In addition, cysteine (Cys) was found to arise in place of selenocysteine in thioredoxin reductase (TR) in NIH 3T3 cells and in mice. An analysis of the selenocysteine biosynthetic pathway demonstrated that replacement of selenide with sulfide in generating the active cysteine donor, thiophosphate, resulted in cysteine being donated to the acceptor molecule, which is likely dehydroalanyl-tRNA[Ser]Sec, yielding Cys-tRNA[Ser]Sec. The identification of the pathways for biosynthesis of selenocysteine and cysteine in mammals is discussed in this chapter.

Selenium (Se) has long been known to be important for male reproduction as severe Se deficiency causes impaired male fertility in livestock, laboratory animals, and humans. In the last decade, the role of Se in male reproduction was elucidated at the molecular level, establishing the roles of specific selenoproteins in this process. Using protein- and isoform-specific knockout mice, it was found that at least two selenoproteins are responsible for the effect of Se: Selenoprotein P, a protein secreted from the liver and serving as the main source of Se for testes, and a mitochondrial form of glutathione peroxidase 4 that has two functions: a peroxidase specific for phospholipid hydroperoxides and a structural component in the midpiece of sperm. Clinical studies further showed that the compromised glutathione peroxidase 4 function in testes is associated with male infertility. In addition, application of X-ray fluorescent microscopy allowed direct visualization of Se distribution in testis and sperm, defining the roles of individual selenoproteins during spermatogenesis. Finally, recent identification of individuals with SBP2 mutations characterized by impaired fertility and azoospermia provided further evidence for importance of Se and selenoproteins in male reproduction.

The classical Trx (thioredoxin) system, composed of TR (Trx reductase), Trx and NADPH, defines a major pathway of cellular thiol-based redox regulation. Three TRs have been identified in mammals: (i) cytosolic TR1, (ii) mitochondrial TR3 and (iii) testes-specific TGR (Trx-glutathione reductase). All three are selenocysteine-containing enzymes with broad substrate specificity in in vitro assays, but which protein substrates are targeted by TRs in vivo is not well understood. In the present study, we used a mechanism-based approach to characterize the molecular targets of TR1. Cytosolic Trx1 was the major target identified in rat and mouse liver, as well as in rat brain and mouse serum. The results suggest that the main function of TR1 is to reduce Trx1. We also found that TR1-based affinity resins provide a convenient tool for specific isolation of Trxs from a variety of biological samples. To better assess the role of TRs in redox homoeostasis, we comparatively analysed TR1- and TR3-knockdown cells. Although cells deficient in TR1 were particularly sensitive to diamide, TR3-knockdown cells were more sensitive to hydrogen peroxide. To further examine the TR1–Trx1 redox pair, we used mice with a liver-specific knockout of selenocysteine tRNA. In this model, selenocysteine insertion into TR1 was blocked, but the truncated form of this protein was not detected. Instead, TR1 and TR3 levels were decreased in the knockout samples. Diminished hepatic TR1 function was associated with elevated Trx1 levels, but this protein was mostly in the oxidized state. Overall, this study provides evidence for the key role of the TR1–Trx1 pair in redox homoeostasis.

The discovery of the genetic code provided one of the basic foundations of modern molecular biology. Most organisms use the same genetic language, but there are also well-documented variations representing codon reassignments within specific groups of organisms (such as ciliates and yeast) or organelles (such as plastids and mitochondria). In addition, duality in codon function is known in the use of AUG in translation initiation and methionine insertion into internal protein positions as well as in the case of selenocysteine and pyrrolysine insertion (encoded by UGA and UAG, respectively) in competition with translation termination. Ambiguous meaning of CUG in coding for serine and leucine is also known. However, a recent study revealed that codons in any position within the open reading frame can serve a dual function and that a change in codon meaning can be achieved by availability of a specific type of RNA stem-loop structure in the 3'-untranslated region. Thus, duality of codon function is a more widely used feature of the genetic code than previously known, and this observation raises the possibility that additional recoding events and additional novel features have evolved in the genetic code.

Mammalian thioredoxin reductases (TRs) are members of the pyridine nucleotide-disulfide oxidoreductase family. The main function of these enzymes is to maintain thioredoxins (Trxs) in the reduced state. The accessibility and high reactivity of selenocysteine in the C-terminal tetrapeptide allows mammalian TRs to couple to a range of substrates from proteins, such as Trx, to small molecules, such as selenite and hydroperoxides. However, identification of physiological substrates of TRs remains a challenge, with new targets identified primarily by testing random candidates in in vitro assays. The reaction mechanism of TRs supports a procedure that could trap substrates in a covalent nonproductive complex with TRs. Accordingly, attachment of TRs to affinity matrices allows isolation and identification of these targets. Application of this method revealed that Trxs are the major targets of TRs and supported efficient isolation of Trx substrates on TR affinity columns. We suggest that this procedure may be used as a general method of affinity isolation of Trxs and other TR substrates.

Translational read-through of the UGA stop codon is an evolutionarily conserved feature that most prominently represents the basis of selenoprotein biosynthesis. It requires a specific cis-acting stem loop control element, termed SECIS, which is located in the 3′-untranslated region of eukaryotic selenoprotein mRNAs. In a search for novel factors underlying the SECIS-directed UGA read-through process, we identified an evolutionary conserved GTPase-activating protein, termed GAPsec. We show that the activity of the Drosophila GAPsec (dGAPsec) is necessary to support SECIS-dependent UGA read-through activity in flies and the mouse homolog mGAPsec in mice tissue culture cells. However, selenoprotein biosynthesis is not impaired in flies that lack dGAPsec activity. The results indicate that GAPsec is part of a novel SECIS-dependent translational read-through system that does not involve selenocysteine incorporation.—Hirosawa-Takamori, M., Ossipov, D., Novoselov, S.V., Turanov, A.A., Zhang, Y., Gladyshev, V.N., Krol, A., Vorbrüggen, G., Jäckle, H. A novel stem loop control element-dependent UGA read-through system without translational selenocysteine incorporation in Drosophila.

Background 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. Results 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 selD 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, Enterococcus faecalis, 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. Conclusion These studies suggest the occurrence of a third Se utilization trait in bacteria and archaea.

Selenocysteine insertion into protein in mammalian cells requires RNA elements in the 3′-untranslated regions (3′-UTRs) of selenoprotein genes. The occurrence of these conserved sequences should make selenoproteins particularly amenable for knockdown/knock-in strategies to examine selenoprotein functions. Herein, we utilized the 3′-UTR of various selenoproteins to knock down their expression using siRNAs and then knock in expression using constructs containing mutations within the target region. Thioredoxin reductase 1 (TR1) knockdown in a mouse kidney cell line resulted in the cells growing about 10% more slowly, being more sensitive to UV radiation, and having increased apoptosis in response to UV than control cells. The knockdown cells transfected with a construct encoding the wild-type TR1 gene and having mutations in the sequences targeted by siRNA restored TR1 expression and catalytic activity, rendered the knockdown cells less sensitive to UV, and protected the cells against apoptosis. We also applied this technique to other selenoproteins, selenophosphate synthetase 2 and glutathione peroxidase 1, and found that mRNA and protein levels were restored following transfection of knockdown cells with the corresponding knock-in constructs. In addition to important new insights into the functions of key mammalian selenoproteins, the data suggest that the RNAi-based knock-in technology could distinguish phenotypes due to off-targeting and provide a new method for examining many of the subtleties of selenoprotein function not available using RNAi technology alone.

Thioredoxin reductase (TR) and thioredoxin (Trx) define a major cellular redox system that maintains cysteine residues in numerous proteins in the reduced state. Both cytosolic (TR1 and Trx1) and mitochondrial (TR3 and Trx2) enzymes are essential in mammals, but the function of the mitochondrial system is less understood. In this study, we characterized subcellular localization of three TR3 forms that are generated by alternative first exon splicing and that differ in their N-terminal sequences. Only one of these forms resides in mitochondria, whereas the two other isoforms are cytosolic. Consistent with this finding, TR3 did not have catalytic preferences for mitochondrial Trx2 versus cytosolic Trx1, both of which could serve as TR3 substrates. Similarly, TR1 was equally active with Trx1, Trx2, or a bacterial Trx. We generated recombinant selenoprotein forms of TR1 and TR3 and found that these enzymes were inhibited by zinc, but not by calcium or cobalt ions. We further developed a proteomic method for identification of targets of TRs in mammalian cells utilizing affinity columns containing recombinant TR3 forms differing in C-terminal sequences. Using this procedure, we found that Trx1 was the major target of TR3 in both rat and mouse liver cytosol. The truncated form of TR3 lacking selenocysteine was particularly efficient in binding Trx1, consistent with the previously observed role of truncated TR1 in apoptosis. Overall, these data establish that the function of TR3 is not limited to its role in Trx2 reduction.