POSTTRANSCRIPTIONAL REGULATION OF ENOS

申请人和其他实验室之前的工作表明，内皮型一氧化氮合酶(ENOS)是通过改变mRNA半衰期在转录后进行调节的。申请人已经证明，在内皮细胞生长过程中，enos mRNA的半衰期大约是细胞静止时的3倍。这似乎是通过内皮型一氧化氮合酶3‘非编码区的新序列与51KDA蛋白之间的相互作用而发生的，该蛋白已被命名为Nos不稳定蛋白(MONSDER)。本研究的第一个目的是克隆和测序它们的蛋白，命名为Nos去稳定剂的消息(MONSDER)。第二个目标是利用四环素调控的逆转录病毒载体系统在内皮细胞中过度表达MONSDER，并确定这对eNOS表达的影响。第三个目标是研究enos mRNA稳定性和核糖体组装之间的关系，并确定MONSDER过表达对这一过程的影响。总体而言，这些研究应该为调节内皮一氧化氮产生的新机制提供洞察力。作为这一病房的一部分，申请者将在药理学系与T.J.墨菲博士一起工作两年，墨菲博士在信使核糖核酸稳定性领域拥有专业知识。随后，他将在埃默里大学心内科获得教员职位和实验室空间，并将继续与墨菲博士和大卫·哈里森博士互动，并得到他们的指导。哈里森博士是他以前的导师，也是一名心脏病学教员。申请者的最终目标是从事基础心血管研究。在获奖期间进行这项研究所获得的经验应该会使瑟尔斯博士成为一名独立的研究员，拥有实现这一目标所需的专业知识和工具。

Previous work, performed by the applicant, and by other laboratories, has shown that the endothelial nitric oxide synthase (eNOS) is regulated post- transcriptionally via changes in mRNA half-life. The applicant has shown that during endothelial cell growth, eNOS mRNA half-life is approximately 3 times longer than when cells are quiescent. This seems to occur via interactions between novel sequences in the Enos 3'utr AND A 51 kDA protein which has been named Message of Nos DestabilizER (MONSDER). The first aim of this research will be to clone and sequence their protein, named Message of Nos DestabilizER (MONSDER). The second aim will be to over-express MONSDER in endothelial cells using a tetracycline- regulated retroviral vector system, and determine the effect of this on eNOS expression. The third aim will be to examine the relationship between eNOS mRNA stability and ribosomal assembly, and determine the effect of MONSDER over-expression on this process. Overall, these studies should provide insight into a novel mechanism of regulation of endothelial nitric oxide production. As a portion of this ward, the applicant will spend two years working in the Department of Pharmacology with Dr. T.J. Murphy, who has expertise in the area of mRNA stability. Subsequently, he will be given a faculty position and laboratory space in the Cardiology Division at Emory, and will continue to have interactions with and guidance from Drs. Murphy and Dr. David Harrison, his previous mentor and a faculty member in Cardiology. The applicant's ultimate goal is to pursue a career in basic cardiovascular research. The experience gained performing this research during the period of this award should permit Dr. Searles to become an independent investigator with the expertise and tools necessary to achieve this goal.