Evaluating Posttranscriptional Regulation of Cytokine Genes
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 wordsmRNA 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.
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