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

Objectives: 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. It has been proposed that 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. This mRNA hijacking mechanism could enable the expression of virally encoded selenoprotein modules, through translation of in-frame UGA stop codons as selenocysteine (Sec). Here, our aim was to assess whether readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, and whether selenium-based mechanisms might be involved. Material and Methods: To assess UGA codon readthrough, we used fluorescence microscopy image analysis and flow cytometry of HEK 293 cells transfected with full length HIV-1 nef gene expression constructs including the 3’-UGA stop codon and a predicted thioredoxin reductase 1 (TXNRD1) antisense region spanning the UGA codon, engineered with a downstream in-frame green fluorescent protein (GFP) reporter gene. These were designed so that GFP can only be expressed by translational recoding of the UGA codon, that is, if the UGA codon is translated as an amino acid or bypassed by ribosomal hopping. To assess readthrough efficiency, appropriate mutant control constructs were used for 100% and 0% readthrough. We used anti-TXNRD1 siRNA to assess the possible role of the proposed antisense interaction in this event, by knockdown of TXNRD1 mRNA levels. Results: UGA stop codon readthrough efficiency for the wild-type nef construct was estimated by flow cytometry to be about 19% (P < 0.0001). siRNA knockdown of TXNRD1 mRNA resulted in a 67% decrease in GFP expression in this system relative to control cells (P < 0.0001), presumably due to reduced availability of the components involved in selenocysteine incorporation for the stop codon readthrough (i.e. the TXNRD1 SECIS element). Addition of 20 nM sodium selenite to the media enhanced stop codon readthrough in the pNefATI1 plasmid construct by >100% (P < 0.0001), that is, more than doubled the amount of readthrough product, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism. Conclusion: Our results show that readthrough of the 3’-terminal UGA codon of nef occurs during translation of HIV-1 nef expression constructs in transfected cells, that this is dependent on selenium concentration, and the presence of TXNRD1 mRNA, supporting the proposed antisense tethering interaction.

The polypyrimidine tract binding protein, PTBP1, regulates selenium homeostasis via the Selenoprotein P 3′ untranslated region

Selenoproteins contain the 21st amino acid, selenocysteine (Sec), which is incorporated at select UGA codons when the encoding mRNA contains a specialized hairpin sequence in its 3′ UTR. This hairpin, the so-called Sec insertion sequence (SECIS) element, is found in all selenoprotein mRNAs, but the sequence surrounding these elements is widely variable and in many cases of considerable length. In order to determine the function of one such SECIS context, we chose to focus on the plasma selenoprotein, SELENOP, that is required to maintain selenium homeostasis. It is unique in that its mRNA contains two SECIS elements that lie in the context of a highly conserved 843-nucleotide 3′ UTR. Prior work has attempted to examine the functions of the SECIS context but none were identified. Here we have used CRISPR/Cas9 genome editing to delete the region between the two SECIS elements. We found that this sequence is required to mediate an increase in SELENOP synthesis under conditions of peroxide stress. Using RNA affinity chromatography, we have identified PTBP1 as the major RNA binding protein that specifically interacts with this region.

The algal selenoproteomes

Background Selenium 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. Results In 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. Conclusions This 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 algal selenoproteomes

Background: Selenium 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. Results: In 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. Conclusions: This 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 algal selenoproteomes

Background:Selenium 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 developing a series of bioinformatics methods to predict and research selenoprotein genes. There are 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, is very limited.Results:In 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 hold these algae selenoprotein genes (www.selenoprotein.com). These genes belong to 42 selenoprotein families, including three novel selenoprotein gene families.Conclusions:This 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 build the whole evolutionary spectrum of selenoproteins for all life.

The algal selenoproteomes

Background: Selenium is an important trace element, and selenocysteine is its predominant form in vivo. The protein containing selenocysteine is selenoprotein, whose special structural features include not only the TGA codon encoding selenocysteine, but also the SECIS element in mRNA and the conservation of selenocysteine flanking region. These special features have led to the development of a series of bioinformatics methods for the prediction and research of selenoprotein genes. There are 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, is very limited. Results: In 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 hold these algae selenoprotein genes (www.selenoprotein.com). These genes belong to 42 selenoprotein families, including three novel selenoprotein gene families. Conclusions: This 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 build the whole evolutionary spectrum of selenoproteins for all life.

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

ABSTRACTSelenoproteins contain the amino acid selenocysteine 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 non-canonical translation mechanism requiring suppression of the UGA stop codon, and a selenocysteine 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 selenocysteine (DMNB-Sec) at the UAG amber stop codon. Recombinantly expressed selenoproteins can be photoactivated in living cells with spatial and temporal control. Using this approach, the native selenoprotein methionine-R-sulfoxide reductase 1 is generated and activated in mammalian cells. The ability to site-specifically introduce selenocysteine directly in mammalian cells, and temporally modulate selenoprotein activity, will aid in the characterization of mammalian selenoprotein function.

Functional Analysis of Genetic Variation in the SECIS Element of Thyroid Hormone Activating Type 2 Deiodinase

Context Thyroid hormone is important for normal brain development. The type 2 deiodinase (D2) controls thyroid hormone action in the brain by activating T4 to T3. The enzymatic activity of D2 depends on the incorporation of selenocysteine for which the selenocysteine-insertion sequence (SECIS) element located in the 3′ untranslated region is indispensable. We hypothesized that mutations in the SECIS element could affect D2 function, resulting in a neurocognitive phenotype. Objective To identify mutations in the SECIS element of DIO2 in patients with intellectual disability and to test their functional consequences. Design, Setting, and Patients The SECIS element of DIO2 was sequenced in 387 patients with unexplained intellectual disability using a predefined pattern of thyroid function tests. SECIS element read-through in wild-type or mutant D2 was quantified by a luciferase reporter system in transfected cells. Functional consequences were assessed by quantifying D2 activity in cell lysate or intact cell metabolism studies. Results Sequence analysis revealed 2 heterozygous mutations: c.5703C&gt;T and c.5730A&gt;T, which were also present in the unaffected family members. The functional evaluation showed that both mutations did not affect D2 enzyme activity in cell lysates or intact cells, although the 5730A&gt;T mutation decreased SECIS element read-through by 75%. In the patient harboring the c.5730A&gt;T variant, whole genome sequencing revealed a pathogenic deletion of the STXBP1 gene. Conclusions We report on two families with mutations in the SECIS element of D2. Although functional analysis showed that nucleotide 5730 is important for normal SECIS element read-through, the two variants did not segregate with a distinct phenotype.

Engineering the elongation factor Tu for efficient selenoprotein synthesis

Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically—using the UAG amber codon—inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O6-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.