Evaluating Posttranscriptional Regulation of Cytokine Genes

  • Bernd Rattenbacher
  • Paul R. BohjanenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 820)


A wide variety of cytokines are necessary for cell–cell communication in multicellular organisms, and cytokine dysregulation has detrimental effects, leading to disease states. Thus, it is a necessity that the expression of cytokines is tightly controlled. Regulation of cytokine gene expression takes place at different levels, including transcriptional and posttranscriptional levels. Ultimately, the steady-state levels of cytokine transcripts are determined by the equilibrium of transcription and degradation of this mRNA. Degradation rates of cytokine mRNAs can be measured in cells by blocking transcription with actinomycin D, harvesting RNA after different time points, and evaluating mRNA levels over time by northern blot. Cis-acting elements that mediate the rapid decay of numerous cytokine transcripts, including AU-rich elements (AREs), are found in the 3′ untranslated region (UTR) of these transcripts. Putative regulatory cis-elements can be cloned into the 3′ UTR of a reporter transcript in order to assess their function in regulating mRNA decay. Cis-elements, such as AREs, regulate cytokine mRNA decay by binding to trans-acting proteins, such as tristetraprolin or HuR. These RNA-binding proteins can be visualized using electromobility shift assays or UV crosslinking assays based on their binding to radioactively labeled RNA sequences. RNA-binding proteins that regulate cytokine mRNA decay can be purified using an RNA affinity method, using their target RNA sequence as the bait. In this chapter, we review the methods for measuring cytokine mRNA decay and methods for characterizing the cis-acting elements and trans-acting factors that regulate cytokine mRNA decay.

Key words

mRNA decay Actinomycin D chase Northern blot RNA–protein interaction EMSA UV crosslinking One-step affinity purification AU-rich element Tristetraprolin HuR 



We thank K. Rattenbacher and I.A. Vlasova for their helpful comments and A-B Shyu for providing β-globin reporter plasmids. This work was supported by NIH grant AI072068.


  1. 1.
    Ogilvie, R.L., Abelson, M., Hau, H.H., Vlasova, I., Blackshear, P.J., Bohjanen, P.R. (2005) Tristetraprolin down-regulates IL-2 gene expression through AU-rich element-mediated mRNA decay. J. Immunol. 174: 953–961.PubMedGoogle Scholar
  2. 2.
    Hamilton, T.A., Novotny, M., Datta, S., Mandal, P., Hartupee, J., Tebo, J., Li, X. (2007) Chemokine and chemoattractant receptor expression: post-transcriptional regulation. J. Leukoc. Biol. 82: 213–219.PubMedCrossRefGoogle Scholar
  3. 3.
    Sandler, H., Stoecklin, G. (2008) Control of mRNA decay by phosphorylation of tristetraprolin. Biochem. Soc. Trans. 36: 491–496.PubMedCrossRefGoogle Scholar
  4. 4.
    Seko, Y., Cole, S., Kasprzak, W., Shapiro, B.A., Ragheb, J.A. (2006) The role of cytokine mRNA stability in the pathogenesis of autoimmune disease. Autoimmun. Rev. 5: 299–305.PubMedCrossRefGoogle Scholar
  5. 5.
    Stoecklin, G., Tenenbaum, S.A., Mayo, T., Chittur, S.V., George, A.D., Baroni, T.E., Blackshear, P.J., Anderson, P. (2008) Genome-wide analysis identifies interleukin-10 mRNA as target of tristetraprolin. J. Biol. Chem. 283: 11689–11699.PubMedCrossRefGoogle Scholar
  6. 6.
    Lam, L.T., Pickeral, O.K., Peng, A. C., Rosenwald, A., Hurt, E.M., Giltnane, J.M., Averett, L.M., Zhao, H., Davis, R.E., Sathyamoorthy, M., Wahl, L.M., Harris, E.D., Mikovits, J.A., Monks, A.P., Hollingshead, M.G., Sausville, E.A., Staudt, L.M. (2001) Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol. Genome Biol. 2: RESEARCH0041.Google Scholar
  7. 7.
    Raghavan, A., Bohjanen, P.R. (2004) Microarray-based analyses of mRNA decay in the regulation of mammalian gene expression. Brief. Funct. Genomic Proteomic 3: 112–124.PubMedCrossRefGoogle Scholar
  8. 8.
    Raghavan, A., Ogilvie, R.L., Reilly, C., Abelson, M.L., Raghavan, S., Vasdewani, J., Krathwohl, M., Bohjanen, P.R. (2002) Genome-wide analysis of mRNA decay in resting and activated primary human T lymphocytes. Nucleic Acids Res. 30: 5529–5538.PubMedCrossRefGoogle Scholar
  9. 9.
    Xu, N., Chen, C.Y., Shyu, A.B. (2001) Versatile role for hnRNP D isoforms in the differential regulation of cytoplasmic mRNA turnover. Mol. Cell. Biol. 21: 6960–6971.PubMedCrossRefGoogle Scholar
  10. 10.
    Hau, H.H., Walsh, R.J., Ogilvie, R.L., Williams, D.A., Reilly, C.S., Bohjanen, P.R. (2007) Tristetraprolin recruits functional mRNA decay complexes to ARE sequences. J. Cell. Biochem. 100: 1477–1492.PubMedCrossRefGoogle Scholar
  11. 11.
    Lykke-Andersen, J., Wagner, E. (2005) Recruitment and activation of mRNA decay enzymes by two ARE-mediated decay activation domains in the proteins TTP and BRF-1. Genes Dev. 19: 351–361.PubMedCrossRefGoogle Scholar
  12. 12.
    Stoecklin, G., Colombi, M., Raineri, I., Leuenberger, S., Mallaun, M., Schmidlin, M., Gross, B., Lu, M., Kitamura, T., Moroni, C. (2002) Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover. Embo J. 21: 4709–4718.PubMedCrossRefGoogle Scholar
  13. 13.
    Bohjanen, P.R., Petryniak, B., June, C.H., Thompson, C.B., Lindsten, T. (1991) An inducible cytoplasmic factor (AU-B) binds selectively to AUUUA multimers in the 3’ untranslated region of lymphokine mRNA. Mol. Cell. Biol. 11: 3288–3295.PubMedGoogle Scholar
  14. 14.
    Bohjanen, P.R., Petryniak, B., June, C.H., Thompson, C.B., Lindsten, T. (1992) AU RNA-binding factors differ in their binding specificities and affinities. J. Biol. Chem. 267: 6302–6309.PubMedGoogle Scholar
  15. 15.
    Fan, X.C., Steitz, J.A. (1998) HNS, a nuclear-cytoplasmic shuttling sequence in HuR. Proc. Natl. Acad. Sci. USA 95: 15293–15298.PubMedCrossRefGoogle Scholar
  16. 16.
    Ford, L.P., Wilusz, J. (1999) An in vitro system using HeLa cytoplasmic extracts that reproduces regulated mRNA stability. Methods 17: 21–27.PubMedCrossRefGoogle Scholar
  17. 17.
    Raghavan, A., Robison, R.L., McNabb, J., Miller, C.R., Williams, D.A., Bohjanen, P.R. (2001) HuA and tristetraprolin are induced following T cell activation and display distinct but overlapping RNA binding specificities. J. Biol. Chem. 276: 47958–47965.PubMedCrossRefGoogle Scholar
  18. 18.
    Shim, J., Karin, M. (2002) The control of mRNA stability in response to extracellular stimuli. Mol. Cells 14: 323–331.PubMedGoogle Scholar
  19. 19.
    Schmidlin, M., Lu, M., Leuenberger, S.A., Stoecklin, G., Mallaun, M., Gross, B., Gherzi, R., Hess, D., Hemmings, B.A., Moroni, C. (2004) The ARE-dependent mRNA-destabilizing activity of BRF1 is regulated by protein kinase B. Embo J. 23: 4760–4769.PubMedCrossRefGoogle Scholar
  20. 20.
    Raghavan, A., Dhalla, M., Bakheet, T., Ogilvie, R.L., Vlasova, I.A., Khabar, K.S., Williams, B.R., Bohjanen, P.R. (2004) Patterns of coordinate down-regulation of ARE-containing transcripts following immune cell activation. Genomics 84: 1002–1013.PubMedCrossRefGoogle Scholar
  21. 21.
    Vlasova, I.A., McNabb, J., Raghavan, A., Reilly, C., Williams, D.A., Bohjanen, K.A., Bohjanen, P.R. (2005) Coordinate stabilization of growth-regulatory transcripts in T cell malignancies. Genomics 86: 159–171.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Microbiology, Center for Infectious Diseases and Microbiology Translational ResearchUniversity of MinnesotaMinneapolisUSA

Personalised recommendations