POST-TRANSCRIPTIONAL CONTROL OF THE HEART AND LUNGS

（改编自申请人的摘要）从基因转录的各种RNA 与心血管和肺部疾病有关的药物在后期受到监管 转录水平。 这些包括β-肾上腺素能受体、β2- 肾上腺素能受体、血管内皮生长因子（VEGF）和 白介素 3 (IL-3) mRNA。 这些转录本富含腺苷/尿苷 其 3' 非翻译区 (3'-UTR) 中的元件 (ARE) 在 调节它们的稳定性和翻译。 ARE 靶向 mRNA 进行快速 衰变，通常通过多聚（A）缩短依赖的衰变途径。 信使RNA ARE 的不稳定、poly(A) 缩短和翻译功能是 由 ARE 结合蛋白 (ARE-BP) 介导。 有趣的是，不合适 控制这些转录本的丰度会导致疾病。 这些 mRNA 的药理学操作需要了解 调节其基因表达的分子机制。 最伟大的 然而，在研究 ARE 如何与 ARE-BP 相互作用以 调节基因表达。 然而，最大的困难在于研究如何 ARE 与 ARE-BP 相互作用来调节基因表达 证明所识别的 ARE-BP 实际上是后处理所必需的 特定转录本的转录调控。 我们开发了一个 系统研究酵母中 ARE 介导的 mRNA 衰减途径。 插入 将肿瘤坏死因子 α (TNFα) ARE 导入酵母的 3'UTR MFA2 mRNA 导致嵌合 mRNA 快速降解。 AUF1的表达， 酿酒酵母中的 ARE-BP 特别影响 这个含有 ARE 的 mRNA 表明酵母系统重演了 在哺乳动物细胞中获得的结果。 本次实验的目的 拨款提案是利用酵母的遗传和分子方面 研究酵母和哺乳动物细胞中 ARE 介导的 mRNA 衰减途径。 具体来说，我们建议：a）研究β- 肾上腺素受体、IL-3 和 VEGF ARE 使用酵母促进 mRNA 周转 酿酒酵母作为模型系统； b) 调查这些 ARE 如何调节 其 mRNA 在哺乳动物细胞和无细胞系统中的稳定性；和c) 开发测定法来表征β-肾上腺素能受体的作用， IL-3 和 VEGF 负责翻译。 这些研究将帮助我们扩展我们的研究范围 关于 ARE 和 ARE-BP 的知识，以及这些相互作用如何控制 与心血管和肺部疾病相关的 mRNA 表达，以及 如癌症和免疫疾病。

(Adapted from applicant's abstract) 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.