MESSENGER RNA METABOLISM IN YEAST

这项建议涉及转录后的一个基本方面， 真核生物中的控制：调节mRNA衰变的分子机制。 至少有三种不同类型的事件触发mRNA的启动 衰变，包括多聚（A）缩短，内切核酸裂解，和 在过早的无义密码子处停止翻译。使用酵母 酿酒酵母作为模型系统，在我实验室的工作表明， 后一种机制，即无义介导的mRNA衰变，需要一个 无义密码子下游的序列（“下游元件”）， 被第二元件（“稳定剂”）的核糖体穿越抑制，和 依赖于几种因子的活性，包括Upf 1 p，Nmd 2 p， Upf 3 p、Nmd 3 p和Dbp 2 p。我们还证明了下游元素是 一个翻译重新起始的位点，一个需要存在 无义密码子和UPF 1、NMD 2和UPF 3基因产物的表达。在这 我试图测试假设，重新启动触发mRNA衰变， 稳定剂控制着特定衰变因子与 核糖体，可能有一个核组件， 主要是细胞质途径。我们将：1）进一步描述 Upf 1 p、Nmd 2 p和Upf 3 p相互之间以及与其他 蛋白质，并测试这种相互作用是否是mRNA衰变所必需的; 2)确定衰变的因子和mRNA序列要求 与重新起始所需的序列一致; 3）确定mRNA序列 稳定器功能所必需的，并确定其对 单个多聚核糖体和mRNP上是否存在特异性因子; 和4）评估Nmd 2 p和其他因素与 细胞核和新合成的mRNA。

This proposal addresses a fundamental aspect of post-transcriptional control in eukaryotes: the molecular mechanisms that regulate mRNA decay. At least three different types of events trigger the initiation of mRNA decay, including poly(A) shortening, endonucleolytic cleavage, and the arrest of translation at a premature nonsense codon. Using the yeast Saccharomyces cerevisiae as a model system, work in my laboratory has shown that the latter mechanism, nonsense-mediated mRNA decay, requires a sequence downstream of the nonsense codon (the "downstream element"), is inhibited by ribosome traversal of a second element (the "stabilizer"), and is dependent on the activity of several factors, including Upf1p, Nmd2p, Upf3p, Nmd3p, and Dbp2p. We have also shown that the downstream element is a site of translational reinitiation, an event that requires the presence of the nonsense codon and the UPF1, NMD2, and UPF3 gene products. In this proposal, I seek to test hypotheses that reinitiation triggers mRNA decay, that the stabilizer governs the association of specific decay factors with the ribosome, and that there may be a nuclear component to this predominantly cytoplasmic pathway. We will: 1) further characterize the interactions of Upf1p, Nmd2p, and Upf3p with each other and with other proteins and test whether such interactions are required for mRNA decay; 2) determine whether the factor and mRNA sequence requirements for decay coincide with those required for reinitiation; 3) define the mRNA sequences essential for stabilizer function and determine their influence on the presence or absence of specific factors on individual polysomes and mRNPs; and 4) assess a possible association of Nmd2p and other factors with the nucleus and with newly synthesized mRNA.