Selenocysteine Insertion Sequence Research Articles

6492 Novel compound heterozygous mutations in the SBP2 gene

Abstract Disclosure: H. Jeong: None. Mutations in the selenocysteine insertion sequence binding protein 2 gene (SECISBP2 also known as SBP2) result in a multisystemic disorder. A 4-year-old Korean girl presented with atypical thyroid function test result, characterized by normal TSH 2.13 μIU/mL, decreased T3 0.51 ng/mL, free T3(FT3) 1.71 (pg/mL), and elevated free thyroxine (FT4) 4.28 ng/dL. Additionally, she exhibited short stature below 2 standard deviations and delayed bone age. She has not yet begun speaking, and her waking ability commenced only recently. She conceived twins through natural pregnancy, but tragically, her twin sibling passed away in utero at 8 weeks of gestation.A targeted next-generation sequencing panel revealed compound heterozygous mutation in SBP2 gene (p.Arg120Ter/p.Arg672Ter) and in TPO gene (p.Glu413Gln).Her serum selenium level was relatively low,, easuring at 6.94 ng/dL. She underwent T3 supplementation and received a daily selenium supplement of 20mcg. Subsequently, her thyroid function tests normalized. Presentation: 6/2/2024

Modular Polymerase Synthesis and Internal Protein Domain Swapping via Dual Opposed Frameshifts in the Ebola Virus L Gene.

Sequence analysis of the Zaire ebolavirus (EBOV) polymerase (L gene) mRNA, using online tools, identified a highly ranked -1 programmed ribosomal frameshift (FS) signal including an ideal slippery sequence heptamer (UUUAAAA), with an overlapping coding region featuring two tandem UGA codons, immediately followed by an RNA region that is the inverse complement (antisense) to a region of the mRNA of the selenoprotein iodothyronine deiodinase II (DIO2). This antisense interaction was confirmed in vitro via electrophoretic gel shift assay, using cDNAs at the EBOV and DIO2 segments. The formation of a duplex between the two mRNAs could trigger the ribosomal frameshift, by mimicking the enhancing role of a pseudoknot structure, while providing access to the selenocysteine insertion sequence (SECIS) element contained in the DIO2 mRNA. This process would allow the -1 frame UGA codons to be recoded as selenocysteine, forming part of a C-terminal module in a low abundance truncated isoform of the viral polymerase, potentially functioning in a redox role. Remarkably, 90 bases downstream of the -1 FS site, an active +1 FS site can be demonstrated, which, via a return to the zero frame, would enable the attachment of the entire C-terminal of the polymerase protein. Using a construct with upstream and downstream reporter genes, spanning a wildtype or mutated viral insert, we show significant +1 ribosomal frameshifting at this site. Acting singly or together, frameshifting at these sites (both of which are highly conserved in EBOV strains) could enable the expression of several modified isoforms of the polymerase. The 3D modeling of the predicted EBOV polymerase FS variants using the AI tool, AlphaFold, reveals a peroxiredoxin-like active site with arginine and threonine residues adjacent to a putative UGA-encoded selenocysteine, located on the back of the polymerase "hand". This module could serve to protect the viral RNA from peroxidative damage.

Severe neurodevelopmental phenotype, diagnostic, and treatment challenges in patients with SECISBP2 deficiency

PurposeDefects in the gene encoding selenocysteine insertion sequence binding protein 2, SECISBP2, result in global impaired selenoprotein synthesis manifesting a complex syndrome with characteristic serum thyroid function tests due to impaired thyroid hormone metabolism. Knowledge about this multisystemic defect remains limited. MethodsGenetic and laboratory investigations were performed in affected members from 6 families presenting with short stature and failure to thrive. ResultsFour probands presented a complex neurodevelopmental profile, including absent speech, autistic features, and seizures. Pediatric neurological evaluation prompted genetic investigations leading to the identification of SECISBP2 variants before knowing the characteristic thyroid tests in 2 cases. Thyroid hormone treatment improved motor development, whereas speech and intellectual impairments persisted. This defect poses great diagnostic and treatment challenges for clinicians, as illustrated by a case that escaped detection for 20 years because SECISBP2 was not included in the neurodevelopmental genetic panel, and his complex thyroid status prompted antithyroid treatment instead. ConclusionThis syndrome uncovers the role of selenoproteins in humans. The severe neurodevelopmental disabilities manifested in 4 patients with SECISBP2 deficiency highlight an additional phenotype in this multisystem disorder. Early diagnosis and treatment are required, and long-term evaluation will determine the full spectrum of manifestations and the impact of therapy.

Engineered mRNA-ribosome fusions for facile biosynthesis of selenoproteins.

Ribosomes are often used in synthetic biology as a tool to produce desired proteins with enhanced properties or entirely new functions. However, repurposing ribosomes for producing designer proteins is challenging due to the limited number of engineering solutions available to alter the natural activity of these enzymes. In this study, we advance ribosome engineering by describing a novel strategy based on functional fusions of ribosomal RNA (rRNA) with messenger RNA (mRNA). Specifically, we create an mRNA-ribosome fusion called RiboU, where the 16S rRNA is covalently attached to selenocysteine insertion sequence (SECIS), a regulatory RNA element found in mRNAs encoding selenoproteins. When SECIS sequences are present in natural mRNAs, they instruct ribosomes to decode UGA codons as selenocysteine (Sec, U) codons instead of interpreting them as stop codons. This enables ribosomes to insert Sec into the growing polypeptide chain at the appropriate site. Our work demonstrates that the SECIS sequence maintains its functionality even when inserted into the ribosome structure. As a result, the engineered ribosomes RiboU interpret UAG codons as Sec codons, allowing easy and site-specific insertion of Sec in a protein of interest with no further modification to the natural machinery of protein synthesis. To validate this approach, we use RiboU ribosomes to produce three functional target selenoproteins in Escherichia coli by site-specifically inserting Sec into the proteins' active sites. Overall, our work demonstrates the feasibility of creating functional mRNA-rRNA fusions as a strategy for ribosome engineering, providing a novel tool for producing Sec-containing proteins in live bacterial cells.

Asgard archaeal selenoproteome reveals a roadmap for the archaea-to-eukaryote transition of selenocysteine incorporation machinery.

Selenocysteine (Sec) is encoded by the UGA codon that normally functions as a stop signal and is specifically incorporated into selenoproteins via a unique recoding mechanism. The translational recoding of UGA as Sec is directed by an unusual RNA structure, the SECIS element. Although archaea and eukaryotes adopt similar Sec encoding machinery, the SECIS elements have no similarities to each other with regard to sequence and structure. We analyzed >400 Asgard archaeal genomes to examine the occurrence of both Sec encoding system and selenoproteins in this archaeal superphylum, the closest prokaryotic relatives of eukaryotes. A comprehensive map of Sec utilization trait has been generated, providing the most detailed understanding of the use of this nonstandard amino acid in Asgard archaea so far. By characterizing the selenoproteomes of all organisms, several selenoprotein-rich phyla and species were identified. Most Asgard archaeal selenoprotein genes possess eukaryotic SECIS-like structures with varying degrees of diversity. Moreover, euryarchaeal SECIS elements might originate from Asgard archaeal SECIS elements via lateral gene transfer, indicating a complex and dynamic scenario of the evolution of SECIS element within archaea. Finally, a roadmap for the transition of eukaryotic SECIS elements from archaea was proposed, and selenophosphate synthetase may serve as a potential intermediate for the generation of ancestral eukaryotic SECIS element. Our results offer new insights into a deeper understanding of the evolution of Sec insertion machinery.

Selenoprotein deficiency disorder predisposes to aortic aneurysm formation

Aortic aneurysms, which may dissect or rupture acutely and be lethal, can be a part of multisystem disorders that have a heritable basis. We report four patients with deficiency of selenocysteine-containing proteins due to selenocysteine Insertion Sequence Binding Protein 2 (SECISBP2) mutations who show early-onset, progressive, aneurysmal dilatation of the ascending aorta due to cystic medial necrosis. Zebrafish and male mice with global or vascular smooth muscle cell (VSMC)-targeted disruption of Secisbp2 respectively show similar aortopathy. Aortas from patients and animal models exhibit raised cellular reactive oxygen species, oxidative DNA damage and VSMC apoptosis. Antioxidant exposure or chelation of iron prevents oxidative damage in patient’s cells and aortopathy in the zebrafish model. Our observations suggest a key role for oxidative stress and cell death, including via ferroptosis, in mediating aortic degeneration.

Cloning and expression of two isoforms of deiodinase gene DIO3 in spotted sea bass (Lateolabrax maculatus)

Type 3 iodothyronine deiodinase (DIO3) is a key enzyme for thyroid hormones, which plays a role in the growth and development of animals. In this study, we identified and cloned two isoforms of DIO3 (LmDIO3a and LmDIO3b) from spotted sea bass (Lateolabrax maculatus). The nucleotide sequences of both LmDIO3a and LmDIO3b had a SECIS (SElenoCysteine Insertion Sequence) element and a T4 domain, and the deduced amino acid sequence similarity was 64.42 %. The mRNAs of both LmDIO3 isoforms were widely distributed in the brain, with high expression levels in the hypothalamus, pituitary, telencephalon and saccus vasculosus (SV). In the non-brain tissues, the expression of LmDIO3a was higher in the retina, heart and spleen, while LmDIO3b was highly expressed in the head kidney and skin. The expression levels of LmDIO3a and LmDIO3b in the hypothalamus and SV were lower in the early stage of gonadal development. However, the expression levels of both in the hypothalamus increased significantly in the stage IV of gonadal development, but in the SV were up-regulated significantly in the stage V. The photoperiod experiment showed that the expressions of them were promoted by short photoperiod (8L:16D) and inhibited by long photoperiod (16L:8D) in the hypothalamus. In the SV, their expression trends were consistent (They firstly decreased, and then increased). In the pituitary, the expression of LmDIO3a presented a trend of up and down, while the expression of LmDIO3b showed an opposite trend. In the gonad, long photoperiod promoted the expression of LmDIO3a, but inhibited the expression of LmDIO3b (P < 0.05). There was no significant change in the expression of LmDIO3a in the retina, but the expression of LmDIO3b was significantly inhibited by short photoperiod and promoted by long photoperiod (P < 0.05). That different tissues have different responses to photoperiod, indicating photoperiod induction may have tissue specificity. Altogether, this study provides a reference for further research on the photoperiod regulation mechanism of DIO3 gene in fish.

ODP478 Effects of peripheral iodothyronine deiodinases on thyroid hormone economy: Insights from the overexpressing mice

Abstract Background In peripheral tissues, triiodothyronine (T3) production and consequent thyroid hormone actions are mainly regulated by iodothyronine deiodinases (DIOs) classified into 3 types: D1, D2, and D3. Although thyroid hormone economy in the DIO-deficient mice and patients carrying genetic variants of DIOs was well clarified, a condition of DIOs upregulation was not enough elucidated. We aimed to investigate effects of peripheral DIOs on thyroid hormone economy and consequent thyroid hormone action using novel DIO-overexpressing mice that were not yet generated. Methods We cloned coding sequences of human DIOs with FLAG-tag and HiBiT-tag sequences into a pcDNA3.1 vector. To obtain full-length proteins, we modified these vectors by cloning selenocysteine insertion sequence of each DIO (SECIS vectors). Western blot analyses and HiBiT lytic assay using HEK293T cells revealed that SECIS vectors expressed full-length proteins with substantial activity. Subsequently, in vivo transfections of pLIVE-based SECIS vectors into male C57BL/6J mice were performed by hydrodynamic gene delivery to generate mice overexpressing DIOs (D1, D2, and D3 mice). Results First, we verified that DIOs were successfully overexpressed in the livers using HiBiT lytic assay as well as RT-PCR. Analyses of multiple organs of D3 mice assured that full-length protein was not significantly expressed in the heart, the kidney, and the skeletal muscle. RT-PCR analyses also suggested that the pituitary glands and the thyroid glands were not transfected. We evaluated thyroid hormone economy of DIO-overexpressing mice compared to Empty mice transfected with pLIVE-Empty vector. D1 mice did not have changes in serum thyroid hormone levels, whereas D2 mice had higher serum fT3 levels. Clearer changes were found in D3 mice. They had hypothyroidism with higher serum rT3 levels. Correspondingly, in their livers, rT3 concentration was higher and T3-responsive genes were downregulated. We elucidated by adding the cohort with levothyroxine (LT4) administration because DIO-overexpressing mice had liver T4 insufficiency probably due to consumption by DIOs. Comparisons between the DIO-overexpressing mice with and without LT4 administration revealed that D2 mice presented upregulation of T3-responsive genes by LT4 administration distinct from Empty mice. Conclusions We generated DIO-overexpressing mice that expressed human DIOs predominantly in the liver. They provided physiological evidence to integrate the phenotypes of the knockout mice and specific clinical situations. D2 mice seemed to recruit T3 into the circulation as well as in the liver. D3 mice had an overt phenotype characterized by consumptive hypothyroidism. In addition, D3 mice are expected to be a novel hypothyroidism model that does not involve disruption of thyroidal hormone secretion. DIO-overexpressing mice can be generated by single injections of plasmid vectors of interest and can contribute to progress in the field of thyroid hormone economy and action. Presentation: No date and time listed

In vivo probing of SECIS-dependent selenocysteine translation in Archaea.

Cotranslational insertion of selenocysteine (Sec) proceeds by recoding UGA to a sense codon. This recoding is governed by the Sec insertion sequence (SECIS) element, an RNA structure on the mRNA, but size, location, structure determinants, and mechanism differ for Bacteria, Eukarya, and Archaea. For Archaea, the structure-function relation of the SECIS is poorly understood, as only rather laborious experimental approaches are established. Furthermore, these methods do not allow for quantitative probing of Sec insertion. In order to overcome these limitations, we engineered bacterial β-lactamase into an archaeal selenoprotein, thereby establishing a reporter system, which correlates enzyme activity to Sec insertion. Using this system, in vivo Sec insertion depending on the availability of selenium and the presence of a SECIS element was assessed in Methanococcus maripaludis Furthermore, a minimal SECIS element required for Sec insertion in M. maripaludis was defined and a conserved structural motif shown to be essential for function. Besides developing a convenient tool for selenium research, converting a bacterial enzyme into an archaeal selenoprotein provides proof of concept that novel selenoproteins can be engineered in Archaea.

Molecular characterization and expression analysis of selenoprotein W gene in rainbow trout (Oncorhynchus mykiss) with dietary selenium levels.

Selenium (Se) plays an essential role in the growth of fish and performs its physiological functions mainly through incorporation into selenoproteins. Our previous studies suggested that the selenoprotein W gene (selenow) is sensitive to changes in dietary Se in rainbow trout. However, the molecular characterization and tissue expression pattern of selenow are still unknown. Here, we revealed the molecular characterization, the tissue expression pattern of rainbow trout selenow and analyzed its response to dietary Se. The open reading frame (ORF) of the selenow gene was composed of 393 base pairs (bp) and encodes a 130-amino-acid protein. The 3' untranslated region (UTR) was 372bp with a selenocysteine insertion sequence (SECIS) element. Remarkably, the rainbow trout selenow gene sequence was longer than those reported for mammals and most other fish. A β1-α1-β2-β3-β4-α2 pattern made up the secondary structure of SELENOW. Furthermore, multiple sequence alignment revealed that rainbow trout SELENOW showed a high level of identity with SELENOW from Salmo salar. In addition, the selenow gene was ubiquitously distributed in 13 tissues with various abundances and was predominantly expressed in muscle and brain. Interestingly, dietary Se significantly increased selenow mRNA expression in muscle. Our results highlight the vital role of selenow in rainbow trout muscle response to dietary Se levels and provide a theoretical basis for studies of selenow.

EIF3 Interacts with Selenoprotein mRNAs.

The synthesis of selenoproteins requires the co-translational recoding of an in-frame UGASec codon. Interactions between the Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2) in the 3′untranslated region (3′UTR) of selenoprotein mRNAs enable the recruitment of the selenocysteine insertion machinery. Several selenoprotein mRNAs undergo unusual cap hypermethylation and are not recognized by the translation initiation factor 4E (eIF4E) but nevertheless translated. The human eukaryotic translation initiation factor 3 (eIF3), composed of 13 subunits (a-m), can selectively recruit several cellular mRNAs and plays roles in specialized translation initiation. Here, we analyzed the ability of eIF3 to interact with selenoprotein mRNAs. By combining ribonucleoprotein immunoprecipitation (RNP IP) in vivo and in vitro with cross-linking experiments, we found interactions between eIF3 and a subgroup of selenoprotein mRNAs. We showed that eIF3 preferentially interacts with hypermethylated capped selenoprotein mRNAs rather than m7G-capped mRNAs. We identified direct contacts between GPx1 mRNA and eIF3 c, d, and e subunits and showed the existence of common interaction patterns for all hypermethylated capped selenoprotein mRNAs. Differential interactions of eIF3 with selenoprotein mRNAs may trigger specific translation pathways independent of eIF4E. eIF3 could represent a new player in the translation regulation and hierarchy of selenoprotein expression.

The selenoprotein P 3' untranslated region is an RNA binding protein platform that fine tunes selenocysteine incorporation.

Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when a specialized hairpin sequence, the Sec insertion sequence (SECIS) element, is present in the 3' UTR. Aside from the SECIS, selenoprotein mRNA 3' UTRs are not conserved between different selenoproteins within a species. In contrast, the 3'-UTR of a given selenoprotein is often conserved across species, which supports the hypothesis that cis-acting elements in the 3'-UTR other than the SECIS exert post-transcriptional control on selenoprotein expression. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, which is required to maintain selenium homeostasis as a selenium transport protein that contains 10 Sec residues. It is unique in that its mRNA contains two SECIS elements in the context of a highly conserved 843-nucleotide 3' UTR. Here we have used RNA affinity chromatography and identified PTBP1 as the major RNA binding protein that specifically interacts with the sequence between the two SECIS elements. We then used CRISPR/Cas9 genome editing to delete two regions surrounding the first SECIS element. We found that these sequences are involved in regulating SELENOP mRNA and protein levels, which are inversely altered as a function of selenium concentrations.

Structure of the mammalian ribosome as it decodes the selenocysteine UGA codon.

The elongation of eukaryotic selenoproteins relies on a poorly understood process of interpreting in-frame UGA stop codons as selenocysteine (Sec). We used cryo-electron microscopy to visualize Sec UGA recoding in mammals. A complex between the noncoding Sec-insertion sequence (SECIS), SECIS-binding protein 2 (SBP2), and 40S ribosomal subunit enables Sec-specific elongation factor eEFSec to deliver Sec. eEFSec and SBP2 do not interact directly but rather deploy their carboxyl-terminal domains to engage with the opposite ends of the SECIS. By using its Lys-rich and carboxyl-terminal segments, the ribosomal protein eS31 simultaneously interacts with Sec-specific transfer RNA (tRNASec) and SBP2, which further stabilizes the assembly. eEFSec is indiscriminate toward l-serine and facilitates its misincorporation at Sec UGA codons. Our results support a fundamentally distinct mechanism of Sec UGA recoding in eukaryotes from that in bacteria.

The expression of essential selenoproteins during development requires SECIS-binding protein 2-like.

The dietary requirement for selenium is based on its incorporation into selenoproteins, which contain the amino acid selenocysteine (Sec). The Sec insertion sequence (SECIS) is an RNA structure found in the 3' UTR of all selenoprotein mRNAs, and it is required to convert in-frame UGA codons from termination to Sec-incorporating codons. SECIS-binding protein 2 (Sbp2) is required for Sec incorporation, but its paralogue, SECIS-binding protein 2-like (Secisbp2l), while conserved, has no known function. Here we determined the relative roles of Sbp2 and Secisbp2l by introducing CRISPR mutations in both genes in zebrafish. By monitoring selenoprotein synthesis with 75Se labeling during embryogenesis, we found that sbp2 -/- embryos still make a select subset of selenoproteins but secisbp2l -/- embryos retain the full complement. Abrogation of both genes completely prevents selenoprotein synthesis and juveniles die at 14 days post fertilization. Embryos lacking Sbp2 are sensitive to oxidative stress and express the stress marker Vtg1. We propose a model where Secisbp2l is required to promote essential selenoprotein synthesis when Sbp2 activity is compromised.

Inherited Disorders of Thyroid Hormone Metabolism Defect Caused by the Dysregulation of Selenoprotein Expression.

Consistent activation and functioning of thyroid hormones are essential to the human body as a whole, especially in controlling the metabolic rate of all organs and systems. Impaired sensitivity to thyroid hormones describes any process that interferes with the effectiveness of thyroid hormones. The genetic origin of inherited thyroid hormone defects and the investigation of genetic defects upon the processing of thyroid hormones are of utmost importance. Impaired sensitivity to thyroid hormone can be categorized into three conditions: thyroid hormone cell membrane transport defect (THCMTD), thyroid hormone metabolism defect (THMD), and thyroid hormone action defect (THAD). THMD is caused by defects in the synthesis and processing of deiodinases that convert the prohormone thyroxine (T4) to the active hormone triiodothyronine (T3). Deiodinase, a selenoprotein, requires unique translation machinery that is collectively composed of the selenocysteine (Sec) insertion sequence (SECIS) elements, Sec-insertion sequence-binding protein 2 (SECISBP2), Sec-specific eukaryotic elongation factor (EEFSEC), and Sec-specific tRNA (TRU-TCA1-1), which leads to the recognition of the UGA codon as a Sec codon for translation into the growing polypeptide. In addition, THMD could be expanded to the defects of enzymes that are involved in thyroid hormone conjugation, such as glucuronidation and sulphation. Paucity of inherited disorders in this category leaves them beyond the scope of this review. This review attempts to specifically explore the genomic causes and effects that result in a significant deficiency of T3 hormones due to inadequate function of deiodinases. Moreover, along with SECISBP2, TRU-TCA1-1, and deiodinase type-1 (DIO1) mutations, this review describes the variants in DIO2 single nucleotide polymorphism (SNP) and thyroid stimulating hormone receptor (TSHR) that result in the reduced activity of DIO2 and subsequent abnormal conversion of T3 from T4. Finally, this review provides additional insight into the general functionality of selenium supplementation and T3/T4 combination treatment in patients with hypothyroidism, suggesting the steps that need to be taken in the future.

Thyroid hormone economy in mice overexpressing iodothyronine deiodinases.

In peripheral tissues, triiodothyronine (T3) production and consequent thyroid hormone actions are mainly regulated by iodothyronine deiodinases (DIOs) classified into 3 types: D1, D2, and D3. We aimed to investigate the effects of peripheral DIOs on thyroid hormone economy independent of the hypothalamus-pituitary-thyroid axis. We cloned coding sequences of human DIOs with FLAG-tag and HiBiT-tag sequences into a pcDNA3.1 vector. To obtain full-length proteins, we modified these vectors by cloning the selenocysteine insertion sequence of each DIO (SECIS vectors). Western blot analyses and HiBiT lytic assay using HEK293T cells revealed that SECIS vectors expressed full-length proteins with substantial activity. Subsequently, in vivo transfections of pLIVE-based SECIS vectors into male C57BL/6J mice were performed by hydrodynamic gene delivery to generate mice overexpressing DIOs predominantly in the liver (D1, D2, and D3mice). After 7days from transfections, mice were analyzed to clarify phenotypes. To summarize, serum thyroid hormone levels did not change in D1mice but D2mice had higher serum free T3levels. D3mice developed hypothyroidism with higher serum reverse T3 (rT3) levels. Transfections with levothyroxine administration suggested that thyroid hormone action was upregulated in D2mice. Our DIO-overexpressing mice provided insights on the physiological properties of upregulated DIOs: D2 augments local thyroid hormone action and recruits T3 into the circulation: D3 decreases circulating T3 and T4levels with elevated rT3, leading to consumptive hypothyroidism. As D3mice are expected to be a novel hypothyroidism model, they can contribute to progress in the field of thyroid hormone economy and action.

SecMS analysis of selenoproteins with selenocysteine insertion sequence and beyond.

Selenocysteine (Sec, U) is the 21st amino acid, and proteins with selenocysteine are defined as selenoproteins. The currently known selenoproteins are all featured by the presence of selenocysteine insertion sequence (SECIS) on their mRNA, and SECIS plays an essential role in the selenocysteine insertion mechanism. However, due to the extremely low occurrences of selenoproteins in a whole proteome (e.g., only 25 selenoproteins in the human proteome) and the low sequence conservation of SECIS, analysis of selenoproteins and discovery of new selenoproteins exclusively on SECIS are intrinsically challenging. To this end, the selenocysteine-specific mass spectrometry (SecMS) and SECIS-independent selenoprotein (SIS) database are developed, showing abilities to profile whole selenoproteomes sensitively and to discover potential new selenoproteins. Here, we detail the SecMS strategy and propose it will advance the exploration for new selenoproteins and functional studies of selenoproteins.

Expressing recombinant selenoproteins using redefinition of a single UAG codon in an RF1-depleted E. coli host strain.

Selenoproteins containing the rare amino acid selenocysteine (Sec), typically being enzymes utilizing the selenium atom of Sec for promoted catalysis of redox reactions, are challenging to obtain at high amounts in pure form. The technical challenges limiting selenoprotein supply derive from intricacies in their translation, necessitating the recoding of a UGA stop codon to a sense codon for Sec. This, in turn, involves the interactions of a Sec-dedicated elongation factor, either directly or indirectly, with a structure in the selenoprotein-encoding mRNA called a SECIS element (Selenocysteine Insertion Sequence), a dedicated tRNA species for Sec with an anticodon for the UGA, and several accessory enzymes and proteins involved in the selenoprotein synthesis. Here, we describe an alternative method for recombinant selenoprotein production using UAG as the Sec codon in a specific strain of E. coli lacking other UAG codons and lacking the release factor RF1 that normally terminates translation at UAG. We also describe how such recombinant selenoproteins can be purified and further analyzed for final Sec contents. The methodology can be used for production of natural selenoproteins in recombinant form as well as for production of synthetic selenoproteins that may be designed to use the unique biophysical properties of Sec for diverse biotechnological applications.

The emerging role of selenium metabolic pathways in cancer: New therapeutic targets for cancer.

Selenium (Se) is incorporated into the body via the selenocysteine (Sec) biosynthesis pathway, which is critical in the synthesis of selenoproteins, such as glutathione peroxidases and thioredoxin reductases. Selenoproteins, which play a key role in several biological processes, including ferroptosis, drug resistance, endoplasmic reticulum stress, and epigenetic processes, are guided by Se uptake. In this review, we critically analyze the molecular mechanisms of Se metabolism and its potential as a therapeutic target for cancer. Sec insertion sequence binding protein 2 (SECISBP2), which is a positive regulator for the expression of selenoproteins, would be a novel prognostic predictor and an alternate target for cancer. We highlight strategies that attempt to develop a novel Se metabolism-based approach to uncover a new metabolic drug target for cancer therapy. Moreover, we expect extensive clinical use of SECISBP2 as a specific biomarker in cancer therapy in the near future. Of note, scientists face additional challenges in conducting successful research, including investigations on anticancer peptides to target SECISBP2 intracellular protein.

Selenium-Dependent Read Through of the Conserved 3'-Terminal UGA Stop Codon of HIV-1 nef.

The HIV-1 nef gene terminates in a 3'-UGA stop codon, which is highly conserved in the main group of HIV-1 subtypes, along with a downstream potential coding region that could extend the nef protein by 33 amino acids, if readthrough of the stop codon occurs. Antisense tethering interactions (ATIs) between a viral mRNA and a host selenoprotein mRNA are a potential viral strategy for the capture of a host selenocysteine insertion sequence (SECIS) element (Taylor et al, 2016) [1]. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine (SeC). Here we show that readthrough of the 3'-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells. This was accomplished via fluorescence microscopy image analysis and flow cytometry of HEK 293 cells, transfected with engineered GFP reporter gene plasmid constructs, in which GFP can only be expressed by translational recoding of the UGA codon. SiRNA knockdown of thioredoxin reductase 1 (TR1) mRNA resulted in a 67% decrease in GFP expression, presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough, thus supporting the proposed ATI. Addition of 20 nM sodium selenite to the media significantly enhanced stop codon readthrough in the pNefATI1 plasmid construct, by >100%, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism.