Determining the effects of epidermal growth factor (EGF) on EGF receptor mRNA stability in KB cells
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Regulation of gene expression occurs at several levels, one being mRNA stability. Cis determinants, such as the poly(A) tail, 3’ and 5’ untranslated regions (UTRs), and coding region sequences along with trans-acting factors contribute significantly to mRNA stability. Our research focuses on the 3’ UTR of epidermal growth factor receptor (EGF-R) mRNA and its potential role as a stability determinant. For this study, a chimeric construct was created containing the well-characterized rabbit 13-globin gene with the EGF-R 3’ UTR replacing the 13-globin 3’ UTR (pBBUIII). A second plasmid (pcDNABBF) differing only in the 3’ UTR being from c-fos was made as a control. Both plasmids are controlled by the serum-induced c-fos promoter. Polysomes from serum- stimulated KB cells transfected with either plasmid will be added to an in vitro mRNA decay assay to determine the half-lives of each transcript. Presently this assay has not yet been performed in our laboratory, but we suspect the EGF-R 3’ UTR will stabilize the 13-globin transcript when compared to the effects of the c-fos 3’ UTR. To study the involvement of nucleases specific to EGF-R mRNA decay, we utilized the in vitro mRNA decay assay using exogenous capped and uncapped EGF-R transcripts. Results suggest a role for the cap and polysomes from cells treated with EGF in increasing EGF-R mRNA stability in vitro. Unraveling the regulatory mechanisms involved in mRNA stability could provide practical ways to control gene expression.