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A Flexible Approach to Studying Post-Transcriptional Gene Regulation in Stably Transfected Mammalian Cells

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Abstract

The study of post-transcriptional regulation is constrained by the technical limitations associated with both transient and stable transfection of chimeric reporter plasmids examining the activity of 3′-UTR cis-acting elements. We report the adaptation of a commercially available system that enables consistent stable integration of chimeric reporter cDNA into a single genomic site in which transcription is induced by tetracycline. Using this system, we demonstrate the tight control afforded by this system and its suitability in mapping the regulatory function of defined cis-acting elements in the human TNF 3′-UTR, as well as the distinct effects of serum starvation on transiently transfected and stably integrated chimeric reporter genes.

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References

  1. Beutler, B., & Cerami, A. (1989). The biology of cachectin/TNF-alpha primary mediator of the host response. Annual Review of Immunology, 7, 625–655.

    Article  CAS  Google Scholar 

  2. Kontoyiannis, D., Pasparakis, M., Pizarro, T. T., Cominelli, F., & Kollias, G. (1999). Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: Implications for joint and gut-associated immunopathologies. Immunity, 10, 387–398.

    Article  CAS  Google Scholar 

  3. Dumitru, C. D., Ceci, J., Tsatsanis, C., Kontoyiannis, D., Stamatakakis, K., Lin, J. H., et al. (2000). TNF-alpha induction by LPS is regulated posttranscriptionally via a Tpl2/ERK-dependent pathway. Cell, 103, 1071–1083.

    Article  CAS  Google Scholar 

  4. Taylor, G. A., Carballo, E., Lee, D. M., Lai, W. S., Thompson, M. J., Patel, D. D., et al. (1996). A pathogenetic role for TNFα in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency. Immunity, 4, 445–454.

    Article  CAS  Google Scholar 

  5. Carballo, E., Lai, W. S., & Blackshear, P. J. (1998). Feedback inhibition of macrophage tumor necrosis factor-a production by tristetraprolin. Science, 281, 100–104.

    Article  Google Scholar 

  6. Rigby, W. F. C., Roy, K., Collins, J., Rigby, S., Connolly, J. E., Bloch, D. B., et al. (2005). Structure/function analysis of TTP: p38 SAPK and LPS stimulation do not alter TTP function. Journal of Immunology, 174, 7883–7893.

    CAS  Google Scholar 

  7. Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C.-Y. A., Shyu, A. B., et al. (1999). The p38 MAP kinase pathways signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich targeted mechanism. EMBO Journal, 18, 4969–4980.

    Article  CAS  Google Scholar 

  8. Vasudevan, S., & Steitz, J. A. (2007). AU-rich-element-mediated upregulation of translation by FXR1 and Argonaute 2. Cell, 128, 1105–1118.

    Article  CAS  Google Scholar 

  9. Brooks, S. A., Connolly, J. E., & Rigby, W. F. C. (2004). The role of mRNA turnover in the regulation of TTP expression: Evidence for an ERK specific, ARE-dependent, autoregulatory pathway. Journal of Immunology, 172, 7263–7271.

    CAS  Google Scholar 

  10. Brooks, S. A., Connolly, J. E., Diegel, R. J., Fava, R. A., & Rigby, W. F. C. (2002). Analysis of tristetraprolin function, expression, and subcellular distribution. Arthritis and Rheumatism, 46, 1362–1370.

    Article  CAS  Google Scholar 

  11. Buxade, M., Parra, J. L., Rousseau, S., Shpiro, N., Marquez, R., Morrice, N., et al. (2005). The Mnks are novel components in the control of TNF alpha biosynthesis and phosphorylate and regulate hnRNP A1. Immunity, 23, 177–189.

    Article  CAS  Google Scholar 

  12. Hamilton, B. J., Genin, A., Cron, R. Q., & Rigby, W. F. C. (2003). Delineation of a novel pathway that regulates CD154 (CD40 Ligand) expression. Molecular and Cellular Biology, 23, 510–525.

    Article  CAS  Google Scholar 

  13. Eszterhas, S. K., Bouhassira, E. E., Martin, D. I., & Fiering, S. (2002). Transcriptional interference by independently regulated genes occurs in any relative arrangement of the genes and is influenced by chromosomal integration position. Molecular and Cellular Biology, 22, 469–479.

    Article  CAS  Google Scholar 

  14. van der Velden, A. W., Voorma, H. O., & Thomas, A. A. (2001). Vector design for optimal protein expression. Biotechniques, 31, 576–580.

    Google Scholar 

  15. Benjamin, D., Colombi, M., Stoecklin, G., & Moroni, C. (2006). A GFP-based assay for monitoring post-transcriptional regulation of ARE-mRNA turnover. Molecular Biosystems, 2, 561–567.

    Article  CAS  Google Scholar 

  16. Soboleski, M. R., Oaks, J., & Halford, W. P. (2005). Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells. FASEB Journal, 19, 440–442.

    CAS  Google Scholar 

  17. Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H. D., et al. (1999). MAPKAP kinase 2 is essential for LPS-induced TNF-alpha biosynthesis. Nature Cell Biology, 1, 94–97.

    Article  CAS  Google Scholar 

  18. Swantek, J. L., Cobb, M. H., & Geppert, T. D. (1997). Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alpha (TNF-alpha) translation: Glucocorticoids inhibit TNF-alpha translation by blocking JNK/SAPK. Molecular and Cellular Biology, 17, 6274–6282.

    CAS  Google Scholar 

  19. Kotlyarov, A., & Gaestel, M. (2002). Is MK2 (mitogen-activated protein kinase-activated protein kinase 2) the key for understanding post-transcriptional regulation of gene expression? Biochemical Society Transactions, 30, 959–963.

    Article  CAS  Google Scholar 

  20. Lasa, M., Brook, M., Saklatvala, J., & Clark, A. R. (2001). Dexamethasone destabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinase p38. Molecular and Cellular Biology, 21, 771–780.

    Article  CAS  Google Scholar 

  21. Holtmann, H., Winzen, R., Holland, P., Eickemeier, S., Hoffmann, E., Wallach, D., et al. (1999). Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cytokine- or stress-activated signal transduction pathways. Molecular and Cellular Biology, 19, 6742–6753.

    CAS  Google Scholar 

  22. Qiu, L., Yoshida, K., Amorim, B. R., Okamura, H., & Haneji, T. (2007). Calyculin A stimulates the expression of TNF-alpha mRNA via phosphorylation of Akt in mouse osteoblastic MC3T3–E1 cells. Molecular and Cellular Endocrinology, 271, 38–44.

    Article  CAS  Google Scholar 

  23. Stoecklin, G., Lu, M., Rattenbacher, B., & Moroni, C. (2003). A constitutive decay element promotes tumor necrosis factor alpha mRNA degradation via an AU-rich element-independent pathway. Molecular and Cellular Biology, 23, 3506–3515.

    Article  CAS  Google Scholar 

  24. Tsien, R. (1998). The green fluorescent protein. Annual Review of Biochemistry, 67, 509–544.

    Article  CAS  Google Scholar 

  25. Yuste, R. (2005). Fluorescence microscopy today. Nature Methods, 2, 902–904.

    Article  CAS  Google Scholar 

  26. Zorio, D. A., & Bentley, D. L. (2004). The link between mRNA processing and transcription: Communication works both ways. Experimental Cell Research, 296, 91–97.

    Article  CAS  Google Scholar 

  27. Carty, S. M., & Greenleaf, A. L. (2002). Hyperphosphorylated C-terminal repeat domain-associating proteins in the nuclear proteome link transcription to DNA/chromatin modification and RNA processing. Molecular and Cellular Proteomics, 1, 598–610.

    Article  CAS  Google Scholar 

  28. Cramer, P., Pesce, C. G., Baralle, F. E., & Kornblihtt, A. R. (1997). Functional association between promoter structure and transcript alternative splicing. Proceedings of the National Academy of Sciences of the United States of America, 94, 11456–11460.

    Article  CAS  Google Scholar 

  29. Kornblihtt, A. R., de la Mata, M., Fededa, J. P., Munoz, M. J., & Nogues, G. (2004). Multiple links between transcription and splicing. RNA, 10, 1489–1498.

    Article  CAS  Google Scholar 

  30. Lee, C., Gyorgy, A., Maric, D., Sadri, N., Schneider, R. J., Barker, J. L., et al. (2008). Members of the NuRD Chromatin Remodeling Complex Interact with AUF1 in developing cortical neurons. Cerebral Cortex, 18, 2909–2919.

    Article  Google Scholar 

  31. Nicolaï, M., Roncato, M. A., Canoy, A. S., Rouquié, D., Sarda, X., Freyssinet, G., et al. (2006). Large-scale analysis of mRNA translation states during sucrose starvation in arabidopsis cells identifies cell proliferation and chromatin structure as targets of translational control. Plant Physiology, 141, 663–673.

    Article  Google Scholar 

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Acknowledgments

This work was supported by the National Institutes of Health (R01AR49834), the COBRE Program (P20RR16437) from of the National Center for Research Resources, the American College of Rheumatology Research and Education "Within Our Reach" program (awarded to W.F.C.R.), the Veterans Administration (Merit Review to R.C.N. and S.A.B.). J.B. was supported by a Resident Research Preceptorship Award from the American College of Rheumatology, Research Education Foundation. M.Z was supported by a NIH training grant T32 AI00736), and a Hitchcock Foundation grant. The authors thank Abigail Fellows for assistance in preparation of the manuscript, and for technical assistance. Drs. Nichols, Botson, and Wang contributed equally to this manuscript. This paper is subject to the NIH Public Access Policy.

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Authors and Affiliations

  1. Veterans Administration Research Service, Veterans Affairs Medical Center, Mailstop 151, 215 North Main Street, White River Junction, VT, 05009-0001, USA

    Ralph C. Nichols & Seth A. Brooks

  2. Departments of Medicine, Dartmouth Medical School, Lebanon, NH, 03756, USA

    Ralph C. Nichols, John Botson, Seth A. Brooks, Moe Zan & William F. C. Rigby

  3. Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH, 03756, USA

    Ralph C. Nichols, John Botson, Xiao Wei Wang, B. JoNell Hamilton, Jane E. Collins, Victoria Uribe, Seth A. Brooks, Moe Zan & William F. C. Rigby

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  1. Ralph C. Nichols
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  2. John Botson
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  3. Xiao Wei Wang
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  4. B. JoNell Hamilton
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  5. Jane E. Collins
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  7. Seth A. Brooks
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  9. William F. C. Rigby
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Correspondence to Ralph C. Nichols.

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Nichols, R.C., Botson, J., Wang, X.W. et al. A Flexible Approach to Studying Post-Transcriptional Gene Regulation in Stably Transfected Mammalian Cells. Mol Biotechnol 48, 210–217 (2011). https://doi.org/10.1007/s12033-010-9360-8

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  • DOI: https://doi.org/10.1007/s12033-010-9360-8

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