NONSENSE-MEDIATED MRNA DECAY PATHWAY

细胞的正常生长和分化是由受调控的 的表达。细胞内的遗传物质。基因调控 表达已被深入研究，并已被证明是 从转录起始到蛋白质的多点调控 稳定性。最近，有一点变得很清楚，即mRNAs的衰减率 差异高达50倍，受到监管，并发挥着重要作用 在控制基因表达方面。我的目标一直是定义这两个 调节mRNA稳定性的顺式和反式作用因子。工作地点： 许多实验室已经证明了翻译的要求 MRNAs降解。理解翻译在信使核糖核酸中的作用 腐烂是这一领域的主要挑战。这是一个明显的例子 关系是观察到无义突变加速了 MRNAs的衰减率是原来的10到20倍。我们对生物多样性的研究 无意义介导的信使核糖核酸衰变途径已经证明：1) 酵母PGK1基因转录本被过早的废话迅速降解 密码子的位置依赖的方式，2)这种衰退需要特定的 无义突变下游序列(初步证据 表明翻译的重新启动是重要的)，3)可能存在 PGK1转录本中的序列元素，当翻译时，呈现 抵抗无义介导的mRNA衰变途径的PGK1转录本 4)至少有一个反式作用因子参与该途径 (PUPF1)。这项提案的目标是：a)确定 顺式作用序列的边界，包括下游和 抗性元件，并通过以下方法确定其功能区 诱变；B)识别基因和突变，i)抑制效应 和ii)参与灭活 无义介导的mR-NA衰变途径 翻译；以及c)开发一种体外信使核糖核酸衰变系统 体内观察到无意义介导的信使核糖核酸衰变途径。一次进去 体外mRNA衰变系统的建立，我们将利用该系统来 确定限速RNA裂解位点，研究生化 无义介导的信使核糖核酸衰变途径及其机制研究 我们将通过基因筛选识别的蛋白质的作用 如上所述。

Proper cellular growth and differentiation is determined by the regulated expression of the. genetic material within the cell. The control of gene expression has been studied intensively and has been demonstrated to be controlled at multiple points from transcriptional initiation to protein stability. Recently, it has become clear that the decay rates of mRNAs vary by as much as fifty-fold, are regulated and play an important role in controlling gene expression. It has been my goal to define both the cis- and trans-acting factors which regulate mRNA stability. Work from many laboratories has demonstrated the requirement for translation in the degradation of mRNAs. Understanding the role of translation in mRNA decay is a major challenge for this field. One clear example of this relationship is the observation that nonsense mutations accelerate the decay rates of mRNAs 10- to 20-fold. Our studies on the nonsense-mediated mRNA decay pathway have demonstrated that: 1) the transcript of yeast PGK1 gene is rapidly degraded by premature nonsense codons in a position-dependent manner, 2) such decay requires specific sequences downstream of the nonsense mutation (preliminary evidence suggests that translational re-initiation is important), 3) there may be a sequence element in the PGK1 transcript that, when translated, renders the PGK1 transcript resistant to the nonsense-mediated mRNA decay pathway and 4) there is at least one trans-acting factor involved in the pathway (pUPF1). The objectives of this proposal are to: A) identify the boundaries of the cis-acting sequences, including both the downstream and resistance elements, and determine their functional regions by mutagenesis; B) identify genes and mutations that i) suppress the effects of the upfl mutation and ii) are involved in inactivating the nonsense-mediated mRNA decay pathway when the resistance element is translated; and C) develop an in vitro mRNA decay system that reflects the nonsense-mediated mRNA decay pathway observed in vivo. Once an in vitro mRNA decay system is established, we will utilize this system to identify the rate limiting RNA cleavage site, investigate the biochemical mechanisms of the nonsense-mediated mRNA decay pathway and identify the role of the proteins that we will be identifying by the genetic screens described above.