Avoidance of Reporter Assay Distortions from Fused Dual Reporters

Overview

Journal RNA

Specialty Molecular Biology

Date 2017 Apr 27

PMID 28442579

Citations 41

Authors

Gary Loughran

Michael T Howard

Andrew E Firth

John F Atkins

Affiliations

Soon will be listed here.

Abstract

Positioning test sequences between fused reporters permits monitoring of both translation levels and framing, before and after the test sequence. Many studies, including those on recoding such as productive ribosomal frameshifting and stop codon readthrough, use distinguishable luciferases or fluorescent proteins as reporters. Occasional distortions, due to test sequence product interference with the individual reporter activities or stabilities, are here shown to be avoidable by the introduction of tandem StopGo sequences (2A) flanking the test sequence. Using this new vector system (pSGDluc), we provide evidence for the use of a 3' stem-loop stimulator for readthrough, but failed to detect the reported readthrough.

Citing Articles

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References

Lindblad-Toh K, Garber M, Zuk O, Lin M, Parker B, Washietl S . A high-resolution map of human evolutionary constraint using 29 mammals. Nature. 2011; 478(7370):476-82. PMC: 3207357. DOI: 10.1038/nature10530. View

Ryan M, Drew J . Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein. EMBO J. 1994; 13(4):928-33. PMC: 394894. DOI: 10.1002/j.1460-2075.1994.tb06337.x. View

Cardno T, Poole E, Mathew S, Graves R, Tate W . A homogeneous cell-based bicistronic fluorescence assay for high-throughput identification of drugs that perturb viral gene recoding and read-through of nonsense stop codons. RNA. 2009; 15(8):1614-21. PMC: 2714747. DOI: 10.1261/rna.1586709. View

Schueren F, Lingner T, George R, Hofhuis J, Dickel C, Gartner J . Peroxisomal lactate dehydrogenase is generated by translational readthrough in mammals. Elife. 2014; 3:e03640. PMC: 4359377. DOI: 10.7554/eLife.03640. View

Jungreis I, Lin M, Spokony R, Chan C, Negre N, Victorsen A . Evidence of abundant stop codon readthrough in Drosophila and other metazoa. Genome Res. 2011; 21(12):2096-113. PMC: 3227100. DOI: 10.1101/gr.119974.110. View

Loughran G, Chou M, Ivanov I, Jungreis I, Kellis M, Kiran A . Evidence of efficient stop codon readthrough in four mammalian genes. Nucleic Acids Res. 2014; 42(14):8928-38. PMC: 4132726. DOI: 10.1093/nar/gku608. View

Grentzmann G, Ingram J, Kelly P, GESTELAND R, Atkins J . A dual-luciferase reporter system for studying recoding signals. RNA. 1998; 4(4):479-86. PMC: 1369633. View

Donnelly M, Gani D, Flint M, Monaghan S, Ryan M . The cleavage activities of aphthovirus and cardiovirus 2A proteins. J Gen Virol. 1997; 78 ( Pt 1):13-21. DOI: 10.1099/0022-1317-78-1-13. View

Atkins J, Wills N, Loughran G, Wu C, Parsawar K, Ryan M . A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go). RNA. 2007; 13(6):803-10. PMC: 1869043. DOI: 10.1261/rna.487907. View

10.

de Felipe P, Luke G, Brown J, Ryan M . Inhibition of 2A-mediated 'cleavage' of certain artificial polyproteins bearing N-terminal signal sequences. Biotechnol J. 2009; 5(2):213-23. PMC: 2978324. DOI: 10.1002/biot.200900134. View

11.

Xin H, Zhong C, Nudleman E, Ferrara N . Evidence for Pro-angiogenic Functions of VEGF-Ax. Cell. 2016; 167(1):275-284.e6. DOI: 10.1016/j.cell.2016.08.054. View

12.

Diao F, White B . A novel approach for directing transgene expression in Drosophila: T2A-Gal4 in-frame fusion. Genetics. 2012; 190(3):1139-44. PMC: 3296248. DOI: 10.1534/genetics.111.136291. View

13.

Donnelly M, Luke G, Mehrotra A, Li X, Hughes L, Gani D . Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. J Gen Virol. 2001; 82(Pt 5):1013-1025. DOI: 10.1099/0022-1317-82-5-1013. View

14.

Doronina V, Wu C, de Felipe P, Sachs M, Ryan M, Brown J . Site-specific release of nascent chains from ribosomes at a sense codon. Mol Cell Biol. 2008; 28(13):4227-39. PMC: 2447138. DOI: 10.1128/MCB.00421-08. View

15.

Sharma P, Yan F, Doronina V, Escuin-Ordinas H, Ryan M, Brown J . 2A peptides provide distinct solutions to driving stop-carry on translational recoding. Nucleic Acids Res. 2011; 40(7):3143-51. PMC: 3326317. DOI: 10.1093/nar/gkr1176. View

16.

Wang Q, Au H, Jan E . Methods for studying IRES-mediated translation of positive-strand RNA viruses. Methods. 2012; 59(2):167-79. DOI: 10.1016/j.ymeth.2012.09.004. View

17.

Fixsen S, Howard M . Processive selenocysteine incorporation during synthesis of eukaryotic selenoproteins. J Mol Biol. 2010; 399(3):385-96. PMC: 2916059. DOI: 10.1016/j.jmb.2010.04.033. View

18.

Eswarappa S, Potdar A, Koch W, Fan Y, Vasu K, Lindner D . Programmed translational readthrough generates antiangiogenic VEGF-Ax. Cell. 2014; 157(7):1605-18. PMC: 4113015. DOI: 10.1016/j.cell.2014.04.033. View

19.

Stiebler A, Freitag J, Schink K, Stehlik T, Tillmann B, Ast J . Ribosomal readthrough at a short UGA stop codon context triggers dual localization of metabolic enzymes in Fungi and animals. PLoS Genet. 2014; 10(10):e1004685. PMC: 4207609. DOI: 10.1371/journal.pgen.1004685. View

20.

Sun H, Lang Z, Zhu L, Huang D . Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation. Plant Cell Rep. 2012; 31(10):1877-87. DOI: 10.1007/s00299-012-1301-5. View