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’-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.

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 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.

scholarly journals L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough

2012 ◽  
Vol 287 (21) ◽  
pp. 17765-17776 ◽  
Author(s):  
Yoshihide Yamaguchi ◽  
Akiko Hayashi ◽  
Celia W. Campagnoni ◽  
Akio Kimura ◽  
Takashi Inuzuka ◽  
...  
Keyword(s):  
Stop Codon ◽  
Stop Codon Readthrough

scholarly journals Modulation of Viral Programmed Ribosomal Frameshifting and Stop Codon Readthrough by the Host Restriction Factor Shiftless

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1230
Author(s):  
Sawsan Napthine ◽  
Chris H. Hill ◽  
Holly C. M. Nugent ◽  
Ian Brierley
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Stop Codon ◽  
Leukemia Virus ◽  
Size Exclusion ◽  
Human Immunodeficiency Virus Replication ◽  
Mobility Shift ◽  
Ribosomal Frameshifting ◽  
Stop Codon Readthrough

The product of the interferon-stimulated gene C19orf66, Shiftless (SHFL), restricts human immunodeficiency virus replication through downregulation of the efficiency of the viral gag/pol frameshifting signal. In this study, we demonstrate that bacterially expressed, purified SHFL can decrease the efficiency of programmed ribosomal frameshifting in vitro at a variety of sites, including the RNA pseudoknot-dependent signals of the coronaviruses IBV, SARS-CoV and SARS-CoV-2, and the protein-dependent stimulators of the cardioviruses EMCV and TMEV. SHFL also reduced the efficiency of stop-codon readthrough at the murine leukemia virus gag/pol signal. Using size-exclusion chromatography, we confirm the binding of the purified protein to mammalian ribosomes in vitro. Finally, through electrophoretic mobility shift assays and mutational analysis, we show that expressed SHFL has strong RNA binding activity that is necessary for full activity in the inhibition of frameshifting, but shows no clear specificity for stimulatory RNA structures.

Identification of a new 2‐amino acid insertion in the integrase coding region of HIV‐1 subtype G isolates

2021 ◽  
Author(s):  
João Pereira‐Vaz ◽  
Pedro Crespo ◽  
Luísa Mocho ◽  
Patrícia Martinho ◽  
Teresa Fidalgo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Coding Region ◽  
Amino Acid Insertion ◽  
Hiv 1

scholarly journals The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code

Open Biology ◽  
2016 ◽  
Vol 6 (11) ◽  
pp. 160246 ◽  
Author(s):  
Julia Hofhuis ◽  
Fabian Schueren ◽  
Christopher Nötzel ◽  
Thomas Lingner ◽  
Jutta Gärtner ◽  
...  
Keyword(s):  
Malate Dehydrogenase ◽  
Stop Codon ◽  
Protein Isoforms ◽  
Translational Readthrough ◽  
Peroxisomal Targeting ◽  
Codon Context ◽  
Targeting Signal ◽  
Stop Codon Readthrough ◽  
Insight Into ◽  
Actual Ratio

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (e x tended protein) to ‘normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases.

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