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mRNA Decay pp 187–204Cite as

Generation of Cell Lines Stably Expressing a Fluorescent Reporter of Nonsense-Mediated mRNA Decay Activity

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1720))

Abstract

Nonsense-mediated mRNA decay (NMD) is a mechanism of mRNA surveillance ubiquitous among eukaryotes. Importantly, NMD not only removes aberrant transcripts with premature stop codons, but also regulates expression of many normal genes. A recently introduced dual-color fluorescent protein-based reporter enables analysis of NMD activity in live cells. In this chapter we describe the method to generate stable transgenic cell lines expressing the splicing-dependent NMD reporter using consecutive steps of lentivirus transduction and Tol2 transposition.

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References

  1. Chang YF, Imam JS, Wilkinson MF (2007) The nonsense-mediated decay RNA surveillance pathway. Annu Rev Biochem 76:51–74

    Article  CAS  PubMed  Google Scholar 

  2. Schweingruber C, Rufener SC, Zünd D, Yamashita A, Mühlemann O (2013) Nonsense-mediated mRNA decay—mechanisms of substrate mRNA recognition and degradation in mammalian cells. Biochim Biophys Acta 1829:612–623

    Article  CAS  PubMed  Google Scholar 

  3. Kertész S, Kerényi Z, Mérai Z, Bartos I, Pálfy T, Barta E, Silhavy D (2006) Both introns and long 3′-UTRs operate as cis-acting elements to trigger nonsense-mediated decay in plants. Nucleic Acids Res 34:6147–6157

    Article  PubMed  PubMed Central  Google Scholar 

  4. Dai Y, Li W, An L (2016) NMD mechanism and the functions of Upf proteins in plant. Plant Cell Rep 35:5–15

    Article  CAS  PubMed  Google Scholar 

  5. Lykke-Andersen S, Jensen TH (2015) Nonsense-mediated mRNA decay: An intricate machinery that shapes transcriptomes. Nat Rev Mol Cell Biol 16:665–677

    Article  CAS  PubMed  Google Scholar 

  6. Bruno IG, Karam R, Huang L, Bhardwaj A, Lou CH, Shum EY, Song HW, Corbett MA, Gifford WD, Gecz J, Pfaff SL, Wilkinson MF (2011) Identification of a microRNA that activates gene expression by repressing nonsense-mediated RNA decay. Mol Cell 42:500–510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Jin Y, Zhang F, Ma Z, Ren Z (2016) MicroRNA 433 regulates nonsense-mediated mRNA decay by targeting SMG5 mRNA. BMC Mol Biol 17:17

    Article  PubMed  PubMed Central  Google Scholar 

  8. Nickless A, Jackson E, Marasa J, Nugent P, Mercer RW, Piwnica-Worms D, You Z (2014) Intracellular calcium regulates nonsense-mediated mRNA decay. Nat Med 20:961–966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Chang L, Li C, Guo T, Wang H, Ma W, Yuan Y, Liu Q, Ye Q, Liu Z (2016) The human RNA surveillance factor UPF1 regulates tumorigenesis by targeting Smad7 in hepatocellular carcinoma. J Exp Clin Cancer Res 35:8

    Article  PubMed  PubMed Central  Google Scholar 

  10. Sanchez G, Bondy-Chorney E, Laframboise J, Paris G, Didillon A, Jasmin BJ, Côté J (2016) A novel role for CARM1 in promoting nonsense-mediated mRNA decay: potential implications for spinal muscular atrophy. Nucleic Acids Res 44:2661–2676

    Article  PubMed  Google Scholar 

  11. Mendell JT, Sharifi NA, Meyers JL, Martinez-Murillo F, Dietz HC (2004) Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat Genet 36:1073–1078

    Article  CAS  PubMed  Google Scholar 

  12. Lykke-Andersen S, Chen Y, Ardal BR, Lilje B, Waage J, Sandelin A, Jensen TH (2014) Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes. Genes Dev 15:2498–2517

    Article  Google Scholar 

  13. Schmidt SA, Foley PL, Jeong DH, Rymarquis LA, Doyle F, Tenenbaum SA, Belasco JG, Green PJ (2015) Identification of SMG6 cleavage sites and a preferred RNA cleavage motif by global analysis of endogenous NMD targets in human cells. Nucleic Acids Res 43:309–323

    Article  CAS  PubMed  Google Scholar 

  14. Boelz S, Neu-Yilik G, Gehring NH, Hentze MW, Kulozik AE (2006) A chemiluminescence-based reporter system to monitor nonsense-mediated mRNA decay. Biochem Biophys Res Commun 349:186–191

    Article  CAS  PubMed  Google Scholar 

  15. Paillusson A, Hirschi N, Vallan C, Azzalin CM, Muhlemann O (2005) A GFP based reporter system to monitor nonsense-mediated mRNA decay. Nucleic Acids Res 33:e54

    Article  PubMed  PubMed Central  Google Scholar 

  16. Pereverzev AP, Gurskaya NG, Ermakova GV, Kudryavtseva EI, Markina NM, Kotlobay AA, Lukyanov SA, Zaraisky AG, Lukyanov KA (2015) Method for quantitative analysis of nonsense-mediated mRNA decay at the single cell level. Sci Rep 5:7729–7738

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Pereverzev AP, Matlashov ME, Staroverov DB, Lukyanov KA, Gurskaya NG (2015) Differences of nonsense-mediated mRNA degradation activity in mammalian cell lines revealed by a fluorescence reporter. Bioorg Khim 41:587–591

    CAS  PubMed  Google Scholar 

  18. Gurskaya NG, Pereverzev AP, Staroverov DB, Markina NM, Lukyanov KA (2016) Analysis of nonsense-mediated mRNA decay at the single-cell level using two fluorescent proteins. Methods Enzymol 572:291–314

    Article  CAS  PubMed  Google Scholar 

  19. Balciunas D, Wangensteen KJ, Wilber A, Bell J, Geurts A, Sivasubbu S, Wang X, Hackett PB, Largaespada DA, McIvor RS, Ekker SC (2006) Harnessing a high cargo-capacity transposon for genetic applications in vertebrates. PLoS Genet 2:e169

    Article  PubMed  PubMed Central  Google Scholar 

  20. McCarron A, Donnelley M, McIntyre C, Parsons D (2016) Challenges of up-scaling lentivirus production and processing. J Biotechnol 240:23–30

    Article  CAS  PubMed  Google Scholar 

  21. Ismail SI, Kingsman SM, Kingsman AJ, Uden M (2000) Split-intron retroviral vectors: enhanced expression with improved safety. J Virol 74:2365–2371

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Barde I, Salmon P, Trono D (2010) Production and titration of lentiviral vectors. Curr Protoc Neurosci 4(21):1–21

    Google Scholar 

  23. Kawakami K, Shima A (1999) Identification of the Tol2 transposase of the medaka fish Oryzias latipes that catalyzes excision of a nonautonomous Tol2 element in zebrafish Danio rerio. Gene 240:239–244

    Article  CAS  PubMed  Google Scholar 

  24. Sato Y, Kasai T, Nakagawa S, Tanabe K, Watanabe T, Kawakami K, Takahashi Y (2007) Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev Biol 305:616–624

    Article  CAS  PubMed  Google Scholar 

  25. Kawakami K, Noda T (2004) Transposition of the Tol2 element, an Ac-like element from the Japanese medaka fish Oryzias latipes, in mouse embryonic stem cells. Genetics 166:895–899

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Koga A, Iida A, Kamiya M, Hayashi R, Hori H, Ishikawa Y, Tachibana A (2003) The medaka fish Tol2 transposable element can undergo excision in human and mouse cells. J Hum Genet 48:231–235

    Article  CAS  PubMed  Google Scholar 

  27. Urasaki A, Morvan G, Kawakami K (2006) Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics 174:639–649

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kawakami K (2007) Tol2: a versatile gene transfer vector in vertebrates. Genome Biol 8(Suppl 1):S7

    Article  PubMed  PubMed Central  Google Scholar 

  29. Nakagawa T, Hoogenraad CC (2011) Lentiviral transgenesis. Methods Mol Biol 693:117–142

    Article  CAS  PubMed  Google Scholar 

  30. Liu YP, Berkhout B (2014) HIV-1-based lentiviral vectors. Methods Mol Biol 1087:273–284

    Article  CAS  PubMed  Google Scholar 

  31. Noensie EN, Dietz HC (2001) A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition. Nat Biotechnol 19:434–439

    Article  CAS  PubMed  Google Scholar 

  32. Usuki F, Yamashita A, Higuchi I, Ohnishi T, Shiraishi T, Osame M, Ohno S (2004) Inhibition of nonsense-mediated mRNA decay rescues the phenotype in Ullrich’s disease. Ann Neurol 55:740–744

    Article  CAS  PubMed  Google Scholar 

  33. Durand S, Cougot N, Mahuteau-Betzer F, Nguyen CH, Grierson DS, Bertrand E, Tazi J, Lejeune F (2007) Inhibition of nonsense-mediated mRNA decay (NMD) by a new chemical molecule reveals the dynamic of NMD factors in P-bodies. J Cell Biol 178:1145–1160

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jozefczuk J, Adjaye J (2011) Quantitative real-time PCR-based analysis of gene expression. Methods Enzymol 500:99–109

    Article  CAS  PubMed  Google Scholar 

  35. Nicholson P, Joncourt R, Mühlemann O (2012) Analysis of nonsense-mediated mRNA decay in mammalian cells. Curr Protoc Cell Biol 27:27.4.1–27.4.61

    Google Scholar 

  36. Pruitt SC, Mielnicki LM, Stewart CC (2004) Analysis of fluorescent protein expressing cells by flow cytometry. Methods Mol Biol 263:239–258

    CAS  PubMed  Google Scholar 

  37. Zeyda M, Borth N, Kunert R, Katinger H (1999) Optimization of sorting conditions for the selection of stable, high-producing mammalian cell lines. Biotechnol Prog 15:953–957

    Article  CAS  PubMed  Google Scholar 

  38. Bovia F, Salmon P, Matthes T, Kvell K, Nguyen TH, Werner-Favre C, Barnet M, Nagy M, Leuba F, Arrighi JF, Piguet V, Trono D, Zubler RH (2003) Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors. Blood 101:1727–1733

    Article  CAS  PubMed  Google Scholar 

  39. Teschendorf C, Warrington KH Jr, Siemann DW, Muzyczka N (2002) Comparison of the EF-1 alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-associated virus. Anticancer Res 22:3325–3330

    CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful to Tatiana Gorodnicheva (Evrogen) for providing pLVT-Katushka. This work was supported by the Russian Science Foundation grant 14-25-00129. The work was partially carried out using equipment provided by the Institute of Bioorganic Chemistry Core Facility (CKP IBCH, supported by Russian Ministry of Education and Science, grant RFMEFI62117X0018).

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Authors and Affiliations

  1. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

    Nadezhda M. Markina, Anton P. Pereverzev, Dmitry B. Staroverov, Konstantin A. Lukyanov & Nadya G. Gurskaya

  2. Nizhny Novgorod State Medical Academy, Nizhny Novgorod, Russia

    Konstantin A. Lukyanov & Nadya G. Gurskaya

Authors
  1. Nadezhda M. Markina
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  2. Anton P. Pereverzev
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  3. Dmitry B. Staroverov
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  4. Konstantin A. Lukyanov
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  5. Nadya G. Gurskaya
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Corresponding author

Correspondence to Nadya G. Gurskaya .

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Editors and Affiliations

  1. Murdoch Children’s Research Institute, The Royal Children’s Hospital, Parkville, Victoria, Australia

    Shireen R. Lamandé

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Markina, N.M., Pereverzev, A.P., Staroverov, D.B., Lukyanov, K.A., Gurskaya, N.G. (2018). Generation of Cell Lines Stably Expressing a Fluorescent Reporter of Nonsense-Mediated mRNA Decay Activity. In: Lamandé, S. (eds) mRNA Decay. Methods in Molecular Biology, vol 1720. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7540-2_14

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  • DOI: https://doi.org/10.1007/978-1-4939-7540-2_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7539-6

  • Online ISBN: 978-1-4939-7540-2

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