POST-TRANSCRIPTIONAL CONTROL OF THE HEART AND LUNGS

(改编自申请人的摘要)从基因转录而来的各种RNA 与心血管和肺部疾病有牵连的人在 转录水平。这些受体包括β-肾上腺素能受体、β-2-受体。 肾上腺素能受体、血管内皮生长因子和 白介素3(IL-3)mRNAs。这些转录本含有丰富的腺苷/尿苷 在其3‘非翻译区(3’-UTR)中起作用的元件(ARE) 调节它们的稳定性和翻译。ARES针对RAPID的mRNA 衰变，通常通过聚(A)缩短依赖的衰变途径。信使核糖核酸 战神的不稳定、多聚(A)缩短和平移功能是 由ARE结合蛋白(ARE-Bps)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mRNAs的药理学操作需要理解 调控其基因表达的分子机制。最伟大的 然而，研究战神如何与ARE-BPS互动是困难的 调控基因表达。然而，最大的困难是研究如何 Ares与ARE-Bps相互作用以调节基因表达是能够 证明确定的ARE-BP实际上是后-BP所需的 转录调控对特定转录本的转录调控我们已经开发出一种 研究酵母中ARE介导的mRNA衰变途径的系统。插入 肿瘤坏死因子α(TNFpha)进入酵母3‘端非编码区 MFA2 m RNA，导致嵌合m RNA迅速降解。AUF1的表达， 酿酒酵母中的一种ARE-BP，专门影响酵母的衰减率 这表明酵母系统重现了 在哺乳动物细胞中获得的结果。这项实验的目标是 GRANT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衰变途径。 具体地说，我们建议：a)调查贝塔如何- 利用酵母促进肾上腺素能受体、IL-3和VEGFARES促进mRNA的转换 酿酒酵母作为模式系统；b)调查这些ARE是如何调节 它们的mRNAs在哺乳动物细胞和无细胞系统中的稳定性；以及c) 开发测试方法来表征β-肾上腺素能受体的作用， IL-3和血管内皮生长因子在翻译上的作用。这些研究将帮助我们扩大我们的 关于ARE和ARE-BPS的知识，以及这些交互如何控制 参与心血管和肺部疾病的mRNAs的表达 就像癌症和免疫紊乱一样。

(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.