Various RNAs transcribed from genes implicated in cardiovascular and pulmonary diseases are regulated at the post- transcriptional level. These include the beta-adrenergic receptor, the beta2- adrenergic receptor, the vascular endothelial growth factor (VEGF) and interleukin-3 (IL-3) mRNAs. These transcripts harbor Adenosine/Uridine-rich elements (AREs) in their 3' untranslated regions (3'-UTR) which play a role in regulating their stability and translation. AREs target mRNAs for rapid decay, usually via a poly (A) shortening-dependent decay pathway. The mRNA destabilizing, poly(A) shortening and translational functions of the AREs are mediated by ARE-binding proteins (ARE-BPs). Interestingly, inappropriate control of the abundance of these transcripts leads to disease. Pharmacological manipulation of these mRNAs requires an understanding of the molecular mechanisms regulating their gene expression. The greatest difficulty, however, in studying how the AREs interact with ARE-BPs to regulate gene expression. The greatest difficulty, however, in studying how the AREs interact with ARE-BPs to regulate gene expression is to be able to demonstrate that the identified ARE-BP is actually required for post- transcriptional regulation of a particular transcript. We have develop a system to investigate the ARE-mediated mRNA decay pathway in yeast. Insertion of the Tumor Necrosis Factor alpha(TNFalpha) ARE into the 3'UTR of the yeast MFA2 mRNA, causes rapid degradation of the chimeric mRNA. Expression of AUF1, an ARE-BP, in the yeast S.cerevisiae specifically affects the decay rate of this ARE-containing mRNA suggesting that the yeast system recapitulates the results obtained in mammalian cells. The goals of the experiments in this grant proposal are to utilize the genetic and molecular aspects of yeast to investigate the ARE-mediated mRNA decay pathway in yeast and mammalian cells. Specifically, we propose to: a) investigate the mechanism of how the beta- adrenergic receptors, IL-3 and VEGF AREs promote mRNA turnover using the yeast S.cerevisiae as a model system; b) investigate how these AREs regulate the stability of their mRNAs in mammalian cells and in cell-free system; and c) develop assays to characterize the effects of the beta-adrenergic receptors, IL-3 and VEGF AREs on translation. These studies will help us expand our knowledge on AREs and ARE-BPs, and how these interactions control the expression of mRNAs involved in cardiovascular and pulmonary diseases, as well as in cancers and immune disorders.
