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

2021 ◽  
Vol 1 ◽  
pp. 1
Author(s):  
Lakmini Premadasa ◽  
Gabrielle Dailey ◽  
Jan A. Ruzicka ◽  
Ethan Will Taylor
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Protein ◽  
Stop Codon ◽  
Mrna Levels ◽  
Coding Region ◽  
Secis Element ◽  
Transfected Cells ◽  
Uga Codon ◽  
Stop Codon Readthrough ◽  
Hiv 1

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.

scholarly journals Selenium-Dependent Readthrough of the Conserved 3’-Terminal UGA Stop Codon of HIV-1 Nef

2020 ◽  
Author(s):  
Lakmini Premadasa ◽  
Gabrielle Dailey ◽  
Jan Ruzicka ◽  
Ethan Taylor
Keyword(s):  
Stop Codon ◽  
Thioredoxin Reductase ◽  
Coding Region ◽  
Hek 293 ◽  
Plasmid Construct ◽  
Uga Codon ◽  
The Media ◽  
Sirna Knockdown ◽  
Stop Codon Readthrough ◽  
Hiv 1

The HIV-1 nef gene terminates in a 3&rsquo;-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&rsquo;-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 &gt;100%, supporting the hypothesis that selenium is involved in the UGA readthrough mechanism.

scholarly journals Homocysteine Down-regulates Cellular Glutathione Peroxidase (GPx1) by Decreasing Translation

2005 ◽  
Vol 280 (16) ◽  
pp. 15518-15525 ◽  
Author(s):  
Diane E. Handy ◽  
Yufeng Zhang ◽  
Joseph Loscalzo
Keyword(s):  
Glutathione Peroxidase ◽  
Untranslated Region ◽  
Stop Codon ◽  
Vascular Dysfunction ◽  
Lipid Peroxides ◽  
Mrna Levels ◽  
Coding Region ◽  
Sv40 Promoter ◽  
Secis Element

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteinein vivoand in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to “read-through” a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3′-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3′-untranslated region of the luciferase gene stimulated read-through by 6–11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p< 0.01) and decreased GPx1 enzyme activity by 45% (p= 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p< 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

scholarly journals Efficient Incorporation of Multiple Selenocysteines Involves an Inefficient Decoding Step Serving as a Potential Translational Checkpoint and RibosomeBottleneck

2006 ◽  
Vol 26 (24) ◽  
pp. 9177-9184 ◽  
Author(s):  
Zoia Stoytcheva ◽  
Rosa M. Tujebajeva ◽  
John W. Harney ◽  
Marla J. Berry
Keyword(s):  
Untranslated Region ◽  
Stop Codon ◽  
Secondary Structures ◽  
Selenoprotein P ◽  
Secis Element ◽  
Nonsense Mediated Decay ◽  
Slowing Down ◽  
Uga Codon ◽  
Sense Codon ◽  
Selenoprotein Mrnas

ABSTRACT Selenocysteine is incorporated into proteins via “recoding” of UGA from a stop codon to a sense codon, a process that requires specific secondary structures in the 3′ untranslated region, termed selenocysteine incorporation sequence (SECIS) elements, and the protein factors that they recruit. Whereas most selenoprotein mRNAs contain a single UGA codon and a single SECIS element, selenoprotein P genes encode multiple UGAs and two SECIS elements. We have identified evolutionary adaptations in selenoprotein P genes that contribute to the efficiency of incorporating multiple selenocysteine residues in this protein. The first is a conserved, inefficiently decoded UGA codon in the N-terminal region, which appears to serve both as a checkpoint for the presence of factors required for selenocysteine incorporation and as a“ bottleneck,” slowing down the progress of elongating ribosomes. The second adaptation involves the presence of introns downstream of this inefficiently decoded UGA which confer the potential for nonsense-mediated decay when factors required for selenocysteine incorporation are limiting. Third, the two SECIS elements in selenoprotein P mRNA function with differing efficiencies, affecting both the rate and the efficiency of decoding different UGAs. The implications for how these factors contribute to the decoding of multiple selenocysteine residues are discussed.

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3′-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5′ of the stop codon, six nucleotides 3′ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3′-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3′-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 and mRNA secondary structure in the 3′-UTR had milder effects. Additionally, we found low readthrough genes to have shorter 3′-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009538
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

334 CONSTRUCTION OF A GENETICALLY ENGINEERED PIG EXPRESSING GREEN FLUORESCENT PROTEIN (GFP)-LABELLED PROTEASOMES

2011 ◽  
Vol 23 (1) ◽  
pp. 263 ◽  
Author(s):  
C. W. O'Gorman ◽  
J. Zhao ◽  
M. S. Samuel ◽  
E. M. Walters ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Cellular Localization ◽  
Fluorescent Protein ◽  
Expressed Sequence Tag ◽  
Stop Codon ◽  
Full Length ◽  
Matrix Attachment Region ◽  
Coding Region ◽  
Public Data ◽  
Fetal Fibroblasts

Proteasomes are large protein complexes involved in protein degradation in eukaryotes and undergo dynamic redistribution between cellular compartments. Characterising the cellular localization of proteasomes at various stages of development and in response to stimuli is of interest. We hypothesised that porcine proteasomes could be visualised in vivo via a ubiquitously expressed transgene fusion comprising a proteasomal subunit and green florescent protein (GFP). The full-length sequence for porcine PSMA-1 was first constructed in silico from public data and was used to retrieve a GenBank expressed sequence tag (EST) sequence that appeared to be full length (accession CO946059; kind gift from R. S. Prather). Primers were designed to remove the stop codon and create homology for cloning with InFusion (Clontech, Palo Alto, CA, USA). The amplimer was inserted into pCAG-CreGFP (Addgene plasmid 13776) in place of the Cre coding region. The resulting plasmid (pKW14) was screened via restriction digest and sequenced for confirmation. This plasmid was confirmed functional in porcine fetal fibroblasts. After removal of the plasmid backbones, pKW14, a G418 resistance cassette (NEO), and the chicken egg white matrix attachment region were co-electroporated into male fetal fibroblasts (10 μg of total DNA, 5:2:2 ratio, respectively). Cells were grown in DMEM with 10% fetal bovine serum (FBS) and selection was initiated 36 h after transfection. Following 12 days of selection at 400 mg L–1 G418, colonies were screened by epifluorescence. Positive colonies were harvested and confirmed transgenic for all 3 input DNAs. Positive colonies were randomly pooled as sets of 3 independent integration events. Embryos were reconstructed via SCNT and transferred to 2 recipients. The fusion rates were 70 and 78%, respectively, with transfer numbers of 120 and 125 fused couplets being transferred into synchronized recipients on Day 0 of heat. Both recipients became pregnant and delivered 2 piglets each on Day 114 by Caesarean section. One live piglet was produced from each litter. Of the 2 live-born piglets, 1 survived beyond Day 3 and continues to be healthy. Transgenic status was verified by PCR. Expression was confirmed by epifluorescence of GFP-labelled proteasomes. This founder will be used to establish a model to evaluated cellular localization of proteasomes in vivo and in culture.

Co-Epression of Two Transgenes in Lentiviral Vector Using Independent Transcriptional Units.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5254-5254
Author(s):  
Friedrich G. Schuening ◽  
Michail M. Zaboikin ◽  
Narasimhachar Srinivasakumar ◽  
Tatiana N. Zaboikina
Keyword(s):  
Flow Cytometry ◽  
Transgene Expression ◽  
Dna Methyltransferase ◽  
Fluorescent Protein ◽  
Lentiviral Vector ◽  
Egfp Expression ◽  
Drug Resistant ◽  
Gfp Expression ◽  
Ic50 Values ◽  
Hiv 1

Abstract There are several gene therapy approaches, which require transfer and co-expression of two transgenes within one target cell. To this end, we have created and tested two-gene expression HIV-1 based vectors, which encode enhanced green fluorescent protein (EGFP) and P144K mutant of canine O6-methylguanine-DNA-methyltransferase (MGMT) transgenes under either the phosphoglycerate kinase gene promoter (PGKp), or the elongation factor 1 alpha promoter (EF1a_p). Eight different configurations of the two transgene expression cassettes were created and tested within the same lentiviral backbone (see Figure). Individual VSV-G pseudotyped vectors stocks were produced and used for infection of canine thyroid adenocarcinoma (CTAC) cells at low multiplicity of infection (MOI = 0.1) to ensure 1 copy of proviral vector per transduced cell. The cells were harvested one week later and an aliquot was assayed for EGFP expression by flow cytometry. Another portion was subjected to selection with O6-benzylguanine (BG, 40 m M for 18 hrs) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, 100 m M for 2 hrs) and kept in culture for additional two weeks to eliminate cells expressing insufficient MGMT. The percentage of GFP positive cells, prior to selection with BG and BCNU, ranged between 0.1 % to 6.6% for the dual-transgene expression cassette encoding HIV-1 vectors. Following selection with BG and BCNU, the percentage of GFP positive cells increased for all vectors with the exception of vector #2 PGEM. Two of the vectors (#1 PMEG and #8 EGMP) demonstrated over 80% GFP positivity after selection. The results of flow cytometry after selection were corroborated by fluorescence microscopy of individual BCNU-resistant colonies. GFP expression was readily detected in drug-resistant colonies transduced with vectors #1 PMEG or #8 EGMP. Weaker GFP expression was detected in drug resistant cells transduced with vectors #3 PMGE, #5 EMPG or #6 EGPM. No significant GFP expression was observed in drug-resistant colonies transduced with vectors #2 PGEM, #4 PGME or #7 EMGP. Drug resistance to BCNU (IC50 values) provided by each of the vectors, was also determined. The data showed that the IC50 values for #1 PMEG and #8 EGMP vectors were 2.4-fold and 4.4-fold, respectively, higher than for mock transduced control cells. The above results indicate that coordinated co-expression of two transgenes using independent expression cassettes is promoter, position and orientation dependent. The data also indicate that two potentially useful vectors (#1 PMEG and #8 EGMP) have been identified for evaluation, ex vivo and in vivo, in the canine model for co-expression of two transgenes.

High-Throughput Flow Cytometry–Based Assay to Identify Apoptosis-Inducing Proteins

2007 ◽  
Vol 12 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Mamatha Sauermann ◽  
Florian Hahne ◽  
Christian Schmidt ◽  
Meher Majety ◽  
Heiko Rosenfelder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Specific Antibody ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Fragmentation ◽  
Protein Fluorescence ◽  
Transfected Cells ◽  
High Throughput Assay

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry—based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers. ( Journal of Biomolecular Screening 2007:510-520)

scholarly journals Cytopathic Killing of Peripheral Blood CD4+ T Lymphocytes by Human Immunodeficiency Virus Type 1 Appears Necrotic rather than Apoptotic and Does Not Require env

2002 ◽  
Vol 76 (10) ◽  
pp. 5082-5093 ◽  
Author(s):  
Michael J. Lenardo ◽  
Sara B. Angleman ◽  
Viengngeun Bounkeua ◽  
Joseph Dimas ◽  
Melody G. Duvall ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
T Lymphocytes ◽  
Peripheral Blood ◽  
Fluorescent Protein ◽  
Jurkat Cells ◽  
Coding Region ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT An important unresolved issue of AIDS pathogenesis is the mechanism of human immunodeficiency virus (HIV)-induced CD4+ T-lymphocyte destruction. We show here that HIV type 1 (HIV-1) exerts a profound cytopathic effect upon peripheral blood CD4+ T lymphocytes that resembles necrosis rather than apoptosis. Necrotic cytopathology was found with both laboratory-adapted strains and primary isolates of HIV-1. We carefully investigated the role of env, which has been previously implicated in HIV cytopathicity. HIV-1 stocks with equivalent infectivity were prepared from constructs with either an intact or mutated env coding region and pseudotyped with the glycoprotein of vesicular stomatitis virus (VSV-G) so that the HIV envelope was not rate-limiting for infection. Infected Jurkat T cells died whether or not env was intact; however, the expression of env accelerated death significantly. The accelerated death was blocked by protease inhibitors, indicating that it was due to reinfection by newly produced virus in env+ cultures. Accordingly, we found no disparity in kinetics in CD4lo Jurkat cells. In highly infected peripheral blood T cells, profound necrosis occurred equivalently with both env+ and env− stocks of HIV-1. We also found that HIV-1 cytopathicity was undiminished by the absence of nef. However, viral stocks made by complementation or packaging of HIV-1 genomes with the natural protein-coding sequences replaced by the green fluorescent protein were highly infectious but not cytopathic. Thus, env can accelerate cell death chiefly as an entry function, but one or more viral functions other than env or nef is essential for necrosis of CD4+ T cells induced by HIV-1.

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