Avoidance of reporter assay distortions from fused dual reporters

Loughran, Gary; Howard, Michael; Firth, Andrew E.; Atkins, John F.

doi:10.1261/rna.061051.117

Cited by 70 publications

(90 citation statements)

References 21 publications

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“…Normal protein synthesis was measured by the expression of full-length FLuc, and SCR was measured by the expression of NLuc when a PTC was inserted at position R154X (UGA). Importantly, by expressing each luciferase from a separate mRNA, we avoid many of the pitfalls that can confound interpretation of bicistronic reporters for studying stop codon readthrough (Loughran, Howard, Firth, & Atkins, 2017;Terenin, Smirnova, Andreev, Dmitriev, & Shatsky, 2017).…”

Section: Aminoglycosides Promote Ptc Readthrough While Simultaneouslymentioning

confidence: 99%

Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

Wangen

Green

2019

Preprint

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Stop codon readthrough (SCR) occurs when the ribosome miscodes at a stop codon. Such readthrough events can be therapeutically desirable when a premature termination codon (PTC) is found in a critical gene. To study SCR in vivo in a genome-wide manner, we treated mammalian cells with aminoglycosides and performed ribosome profiling. We find that in addition to stimulating readthrough of PTCs, aminoglycosides stimulate readthrough of normal termination codons (NTCs) genome-wide. Stop codon identity, the nucleotide following the stop codon, and the surrounding mRNA sequence context all influence the likelihood of SCR. In comparison to NTCs, downstream stop codons in 3′UTRs are recognized less efficiently by ribosomes, suggesting that targeting of critical stop codons for readthrough may be achievable without general disruption of translation termination. Finally, we find that G418 treatment globally alters gene expression with substantial effects on translation of histone genes, selenoprotein genes, and S-adenosylmethionine decarboxylase (AMD1).

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Section: Aminoglycosides Promote Ptc Readthrough While Simultaneouslymentioning

confidence: 99%

Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

Wangen

Green

2019

Preprint

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“…According to our model, an increased readthrough efficiency should accelerate formation of the queue that results in reducing reporter expression. To test this, we measured the activity of R Luc in constructs containing the AMD1 tail with R Luc stop codons in contexts known to permit varying levels of readthrough 14,29 . To measure readthrough efficiencies of the different stop codon contexts, it was necessary to first eliminate the AMD1 tail effect by removing the last 50 codons (Fig.…”

Section: Adometdc Catalyses the Decarboxylation Of S-adenosylmethionimentioning

confidence: 99%

“…The amplicons were generated by standard one or multiple PCR reactions. Plasmids used in this study include pEGFP-C1 (Clontech), pcDNA3-HA (Invitrogen), pDluc 27,28 and pSDLuc 29 . pDluc was modified such that the second luciferase reporter (firefly) is expressed under the control of the EMCV IRES.…”

Section: Cloningmentioning

confidence: 99%

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AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation

Yordanova

Loughran

Zhdanov

et al. 2018

Nature

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“…Translation recoding rates were monitored using pJD2257based Dual-Luciferase vectors, a modification of pSGDluc (61) in which a duplicate BamHI and SalI restriction site was eliminated, leaving unique sites located between the Renilla and firefly luciferase coding sequences. DNA sequences harboring the CHIKV recoding signals containing 5Ј and 3Ј overlapping sequences with pJD2257 were designed in silico and synthesized by Genewiz (Gaithersburg, MD) (Table S1).…”

Section: Dual Reporter Plasmid Constructionmentioning

confidence: 99%

Functional and structural characterization of the chikungunya virus translational recoding signals

Kendra¹,

Advani

Chen³

et al. 2018

Journal of Biological Chemistry

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Climate change and human globalization have spurred the rapid spread of mosquito-borne diseases to naïve populations. One such emerging virus of public health concern is chikungunya virus (CHIKV), a member of the Togaviridae family, genus Alphavirus. CHIKV pathogenesis is predominately characterized by acute febrile symptoms and severe arthralgia, which can persist in the host long after viral clearance. CHIKV has also been implicated in cases of acute encephalomyelitis, and its vertical transmission has been reported. Currently, no FDA-approved treatments exist for this virus. Recoding elements help expand the coding capacity in many viruses and therefore represent potential therapeutic targets in antiviral treatments. Here, we report the molecular and structural characterization of two CHIKV translational recoding signals: a termination codon read-through (TCR) element located between the nonstructural protein 3 and 4 genes and a programmed ؊1 ribosomal frameshift (؊1 PRF) signal located toward the 3 end of the CHIKV 6K gene. Using Dual-Luciferase and immunoblot assays in HEK293T and U87MG mammalian cell lines, we validated and genetically characterized efficient TCR and ؊1 PRF. Analyses of RNA chemical modification data with selective 2-hydroxyl acylation and primer extension (SHAPE) assays revealed that CHIKV ؊1 PRF is stimulated by a tightly structured, triple-stem hairpin element, consistent with previous observations in alphaviruses, and that the TCR signal is composed of a single large multibulged hairpin element. These findings illuminate the roles of RNA structure in translational recoding and provide critical information relevant for design of live-attenuated vaccines against CHIKV and related viruses.

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Avoidance of reporter assay distortions from fused dual reporters

Cited by 70 publications

References 21 publications

Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

Stop Codon Context Influences Genome-Wide Stimulation of Termination Codon Readthrough by Aminoglycosides

AMD1 mRNA employs ribosome stalling as a mechanism for molecular memory formation

Functional and structural characterization of the chikungunya virus translational recoding signals

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