Chapter 3. Assays of adenylate uridylate-rich element-mediated mRNA decay in cells.

The abundance of a cytoplasmic mRNA in eukaryotes often determines the level of the encoded protein product. The rates at which an mRNA is synthesized, exported, and degraded collectively contribute to its abundance in all cell types. Numerous mRNAs, particularly those encoding structural proteins, are very stable, with half-lives in the order of many hours. In contrast, mRNAs encoding regulatory proteins, including oncoproteins, cytokines, and signaling proteins, are relatively unstable with half-lives of an hour or less. As a result, modest changes in their decay rates affect their levels over a relatively short time period. This is particularly important to ensure rapid responses to extracellular signaling events. Messenger RNAs often harbor sequence elements that dictate their degradation rates. Adenylate uridylate (A+U)-rich elements (AREs), first identified in 1986, are perhaps the best characterized sequences that promote rapid mRNA degradation. These elements, localized within 3'-untranslated regions, sometimes contain AUUUA pentamers within an overall U-rich sequence, but this does not always define a bona fide ARE. Thus, experimental validation is essential before bestowing upon a suspected A+U-rich sequence the title of "ARE." This chapter describes a reporter gene system that permits quantitative assessment of the effects of candidate A+U-rich sequences on mRNA half-life. This system employs tetracycline-controlled transcriptional silencing of the reporter gene, isolation of total-cell RNA at selected time points, quantitative reverse transcriptase polymerase chain reaction analysis of reporter mRNA levels, and nonlinear regression analysis of mRNA level as a function of time to quantitatively define parameters describing mRNA decay kinetics. Finally, this chapter describes more specialized assays to characterize ARE-mediated mRNA decay pathways, including deadenylation, and discusses decapping.