scholarly journals Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES

2021 ◽  
Vol 5 ◽  
pp. 221
Author(s):  
Martina M. Yordanova ◽  
Gary Loughran ◽  
John F. Atkins ◽  
Pavel V. Baranov
Keyword(s):  
Stop Codon ◽  
Mrna Translation ◽  
Inhibitory Effect ◽  
Expression Vectors ◽  
Reporter System ◽  
Reporter Expression ◽  
Reading Frame ◽  
Reporter Activity ◽  
Dual Reporter ◽  
Stop Codon Readthrough

Background: Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (ORF) that we termed AMD1. To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. To test this hypothesis, we placed the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression driven by the EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: We employ dual luciferase assays, western blotting and RT-qPCR to explore the effects of test sequences downstream to the reporter stop codon on its expression in dual and monocistronic reporter vectors. Results:  With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter’s ORF. In a monocistronic vector without an IRES, the test sequences had distinct effects which were reflective of their properties e.g. AMD1 tail inhibitory effect. We further show with RT-qPCR that the EMCV IRES driven expression of a reporter is an accurate proxy of reporter RNA levels.  Conclusions: While our findings provide little new information regarding the functional role of AMD1 tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of read through permissive sequences and of IRES elements, though these require a separate investigation.

scholarly journals Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES

2020 ◽  
Vol 5 ◽  
pp. 221
Author(s):  
Martina M. Yordanova ◽  
Gary Loughran ◽  
John F. Atkins ◽  
Pavel V. Baranov
Keyword(s):  
Stop Codon ◽  
Mrna Translation ◽  
Luciferase Assay ◽  
Expression Vectors ◽  
Reporter System ◽  
Reporter Expression ◽  
Reading Frame ◽  
Reporter Activity ◽  
Dual Reporter ◽  
Stop Codon Readthrough

Background: Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (AMD1 tail). To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. To test this hypothesis, we placed the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression governed by EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: Here we employ dual luciferase assay and western blotting to explore the effects of AMD1 tail and control sequences on reporter expression in dual and monocistronic reporter vectors.       Results:  With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter’s ORF. The decreased reporter activity that appears to be induced by the readthrough sequence occurs only in reporters containing EMCV IRES. Monocistronic reporters with the same readthrough context sequence exhibit only a modest reduction in reporter activity. Furthermore, in monocistronic vectors, the disproportionate reduction of reporter levels greatly increased when AMD1 tail was translated as a result of readthrough. Such readthrough-mediated reduction was not observed when AMD1 tail was substituted with unrelated sequences in agreement with our original hypothesis. Conclusions: While our findings provide little new information regarding the functional role of AMD1 tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of readthrough permissive sequences and of IRES elements, though these require a separate investigation.

scholarly journals Growth phase dependent stop codon readthrough and shift of translation reading frame inEscherichia coli

FEBS Letters ◽  
1998 ◽  
Vol 421 (3) ◽  
pp. 237-242 ◽  
Author(s):  
Anne-Marie K Wenthzel ◽  
Martin Stancek ◽  
Leif A Isaksson
Keyword(s):  
Growth Phase ◽  
Stop Codon ◽  
Inescherichia Coli ◽  
Reading Frame ◽  
Stop Codon Readthrough

scholarly journals Identification of compounds enhancing Aqp4 stop codon readthrough

2020 ◽  
Author(s):  
D. Sapkota ◽  
J.D. Dougherty
Keyword(s):  
Stop Codon ◽  
Neurological Diseases ◽  
Water Channel ◽  
Luciferase Reporter ◽  
Reporter System ◽  
Experimental Systems ◽  
Reporter Systems ◽  
Approved Drugs ◽  
Pharmacologically Active ◽  
Stop Codon Readthrough

AbstractAn unusual stop codon readthrough event generates a conserved C-terminally elongated variant of the water channel protein Aquaporin 4 (AQP4). In the brain, AQP4 is astrocyte-specific, required for normal functioning of the glymphatic system, and involved in the clearance of the Alzheimer’s associated protein Amyloid beta. Further, the readthrough variant is localized exclusively perivascularly, and the perivascular pool of AQP4 is reduced in Alzheimer’s and several other neurological diseases. However, there are currently no means of increasing or restoring the perivascular pool AQP4. Here we identify a compound that can enhance Aqp4 stop codon readthrough. We screened 2600 compounds, mostly approved drugs and pharmacologically active natural compounds, using a luciferase reporter system. 28 candidate lead compounds were then subjected to a variety of secondary screening steps using orthogonal reporter systems and characterizing dose-response activities. Finally, we tested the top compounds’ abilities to generate readthrough of the endogenous Aqp4 transcript, identifying Apigenin as an enhancer of this biological phenomenon. This compound can allow modulation of readthrough in experimental systems, mechanistic studies of programmed readthrough, and suggests the potential for modulating Alzheimer’s disease through pharmacological enhancement of perivascular AQP4.

Translational termination–re-initiation in viral systems

2008 ◽  
Vol 36 (4) ◽  
pp. 717-722 ◽  
Author(s):  
Michael L. Powell ◽  
T. David K. Brown ◽  
Ian Brierley
Keyword(s):  
Translational Control ◽  
Stop Codon ◽  
Short Review ◽  
Start Codon ◽  
Influenza B ◽  
Control Mechanisms ◽  
Viral Mrnas ◽  
Reading Frame ◽  
Translational Termination ◽  
Stop Codon Readthrough

Viruses have evolved a number of translational control mechanisms to regulate the levels of expression of viral proteins on polycistronic mRNAs, including programmed ribosomal frameshifting and stop codon readthrough. More recently, another unusual mechanism has been described, that of termination-dependent re-initiation (also known as stop–start). Here, the AUG start codon of a 3′ ORF (open reading frame) is proximal to the termination codon of a uORF (upstream ORF), and expression of the two ORFs is coupled. For example, segment 7 mRNA of influenza B is bicistronic, and the stop codon of the M1 ORF and the start codon of the BM2 ORF overlap in the pentanucleotide UAAUG (stop codon of M1 is shown in boldface and start codon of BM2 is underlined). This short review aims to provide some insights into how this translational coupling process is regulated within different viral systems and to highlight some of the differences in the mechanism of re-initiation on prokaryotic, eukaryotic and viral mRNAs.

scholarly journals Translation inhibitory elements from Hoxa3 and Hoxa11 mRNAs use uORFs for translation inhibition

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Fatima Alghoul ◽  
Schaeffer Laure ◽  
Gilbert Eriani ◽  
Franck Martin
Keyword(s):  
Molecular Mechanisms ◽  
Stop Codon ◽  
Mrna Translation ◽  
Upstream Open Reading Frame ◽  
Initiation Factor ◽  
Entry Site ◽  
80S Ribosome ◽  
Stem Loop ◽  
Reading Frame ◽  
Translation Inhibition

During embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an internal ribosome entry site (IRES) enables cap-independent translation. The second regulon is a translation inhibitory element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of mouse Hoxa3 and Hoxa11 TIEs. Both TIEs possess an upstream open reading frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In Hoxa3 TIE, we identify an uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of Hoxa11 TIE is different, it also contains an uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The ‘start-stop’ sequence is species-specific and in mice, is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hoxa11 main ORF.

scholarly journals Translation Inhibitory Elements from Hox a3 and a11 mRNAs use uORFs for translation inhibition

2021 ◽  
Author(s):  
Fatima Alghoul ◽  
Laure Schaeffer ◽  
Gilbert Eriani ◽  
Franck Martin
Keyword(s):  
Molecular Mechanisms ◽  
Stop Codon ◽  
Mrna Translation ◽  
Upstream Open Reading Frame ◽  
Initiation Factor ◽  
Entry Site ◽  
80S Ribosome ◽  
Stem Loop ◽  
Reading Frame ◽  
Translation Inhibition

AbstractDuring embryogenesis, Hox mRNA translation is tightly regulated by a sophisticated molecular mechanism that combines two RNA regulons located in their 5’UTR. First, an Internal Ribosome Entry Site (IRES) enables cap-independent translation. The second regulon is a Translation Inhibitory Element or TIE, which ensures concomitant cap-dependent translation inhibition. In this study, we deciphered the molecular mechanisms of Hox a3 and a11 TIE elements. Both TIEs possess an upstream Open Reading Frame (uORF) that is critical to inhibit cap-dependent translation. However, the molecular mechanisms used are different. In TIE a3, we identify a uORF which inhibits cap-dependent translation and we show the requirement of the non-canonical initiation factor eIF2D for this process. The mode of action of TIE a11 is different, it also contains a uORF but it is a minimal uORF formed by an uAUG followed immediately by a stop codon, namely a ‘start-stop’. The a11 ‘start-stop’ sequence is located upstream of a highly stable stem loop structure which stalls the 80S ribosome and thereby inhibits cap-dependent translation of Hox a11 main ORF.

scholarly journals New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

2014 ◽  
Vol 42 (15) ◽  
pp. 10061-10072 ◽  
Author(s):  
Sandra Blanchet ◽  
David Cornu ◽  
Manuela Argentini ◽  
Olivier Namy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Stop Codon ◽  
Reporter System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Systematic Analysis ◽  
Stop Codons ◽  
Nucleotide Context ◽  
Stop Codon Readthrough

AbstractStop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNAGln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.

scholarly journals Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

PLoS ONE ◽  
2009 ◽  
Vol 4 (10) ◽  
pp. e7354 ◽  
Author(s):  
Nga T. Lao ◽  
Alan P. Maloney ◽  
John F. Atkins ◽  
Tony A. Kavanagh
Keyword(s):  
Reporter Gene ◽  
Stop Codon ◽  
Dual Reporter ◽  
Stop Codon Readthrough

scholarly journals Tissue-specific dynamic codon redefinition in Drosophila

2020 ◽  
Author(s):  
Andrew M. Hudson ◽  
Gary Loughran ◽  
Nicholas L. Szabo ◽  
Norma M. Wills ◽  
John F. Atkins ◽  
...  
Keyword(s):  
High Efficiency ◽  
Stop Codon ◽  
Open Reading Frame ◽  
Stem Loop ◽  
Tissue Specific ◽  
Reading Frame ◽  
Cis Acting ◽  
A Subunit ◽  
Specific Regulation ◽  
Stop Codon Readthrough

Stop codon readthrough during translation occurs in many eukaryotes, including Drosophila, yeast, and humans. Recoding of UGA, UAG or UAA to specify an amino acid allows the ribosome to synthesize C-terminally extended proteins. We previously found evidence for tissue-specific regulation of stop codon readthrough in decoding the Drosophila kelch gene, whose first open reading frame (ORF1) encodes a subunit of a Cullin3-RING ubiquitin ligase. Here, we show that the efficiency of kelch readthrough varies markedly by tissue. Immunoblotting for Kelch ORF1 protein revealed high levels of the readthrough product in lysates of larval and adult central nervous system (CNS) tissue and larval imaginal discs. A sensitive reporter of kelch readthrough inserted after the second kelch open reading frame (ORF2) directly detected synthesis of Kelch readthrough product in these tissues. To analyze the role of cis-acting sequences in regulating kelch readthrough, we used cDNA reporters to measure readthrough in both transfected human cells and transgenic Drosophila. Results from a truncation series suggest that a predicted mRNA stem-loop 3’ of the ORF1 stop codon stimulates high-efficiency readthrough. Expression of cDNA reporters using cell type-specific Gal4 drivers revealed that CNS readthrough is restricted to neurons. Finally, we show that high-effficiency readthrough in the CNS is common in Drosophila, raising the possibility that the neuronal proteome includes many proteins with conserved C-terminal extensions. This work provides new evidence for a remarkable degree of tissue- and cell-specific dynamic stop codon redefinition in Drosophila.

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