MRNA DECAPPING IN YEAST

我们工作的长期目标是了解控制mRNA稳定性的分子机制。尽管最近的许多观察都强调了mRNA转换在转录后基因调控中的重要性，但人们对这一过程是如何被调节从而导致mRNA衰减率的大范围变化知之甚少。我们已经描述了酵母中mRNA衰变的主要途径，其中聚(A)尾缩短触发脱帽，导致5‘到3’外核溶解。脱帽是该机制中的关键步骤，因为它是诱导mRNA降解的步骤，并且是许多控制输入的位点。例如，聚(A)尾部对mRNA衰变的主要影响是通过抑制脱帽。此外，调节mRNA衰减率的特定序列可以通过影响脱帽率来实现。此外，一种降解异常转录本的特殊衰变途径可以触发极快的mRNA脱冠，而不依赖于poly(a)尾巴缩短。鉴于这一重要性，我们将重点放在理解mRNA脱帽的机制上。在过去的资助期内，我们发现了许多组成mRNA脱帽机制的相互作用蛋白，包括脱帽酶（Dcp1p），所需的正调节因子（Dcp2p）和Lsm蛋白复合物，它们与snRNPs的核心成分Sm蛋白相关。在这项资助中，我们将使用遗传和生化方法的组合来确定这些蛋白质在mRNA脱帽中的功能。该分析将为酵母和更复杂的真核生物的mRNA周转机制提供见解。具体的实验目的是：1 .确定Dcp2蛋白的功能。2。研究mRNA衰变蛋白之间相互作用的意义。3。研究LSM复合体与mRNA相互作用的性质和意义。Iv.检查LSM蛋白是否在翻译中起作用。

The long term goal of our work is to understand the molecular mechanisms that control mRNA stability. Despite many recent observations that emphasize the importance of mRNA turnover in post- transcriptional gene regulation, little is known about how this process modulated to give rise to the broad range of mRNA decay rates. We have described the major pathway of mRNA decay in yeast wherein poly(A) tail shortening triggers decapping, leading to 5' to 3' exonucleolysis. Decapping is a key step in this mechanism because it is the step that induces degradation of the mRNA, and it is the site of numerous control inputs. For example the major effect of the poly(A) tail on mRNA decay is through an inhibition of decapping. I addition, specific sequences that modulate mRNA decay rate can do so by affecting the rates of decapping. Moreover, a specialized decay pathway that degrades aberrant transcripts works triggering extremely rapid mRNA decapping independent of poly(A) tail shortening. Given this importance, we have focused on understanding the mechanisms of mRNA decapping. In the past funding period, we identified a number of interacting proteins that comprise the mRNA decapping machinery including the decapping enzyme (Dcp1p), a required positive regulator (Dcp2p), and a complex of Lsm proteins, which are related to the Sm proteins that are core components of snRNPs. In this grant, we will use a combination of genetic and biochemical approaches to determine the functions of these proteins in mRNA decapping. This analysis will provide insight into the mechanisms of mRNA turnover, both in yeast and in more complex eukaryotes. The specific experimental aims are: I. TO DETERMINE THE FUNCTION OF THE Dcp2 PROTEIN. II. TO EXAMINE THE SIGNIFICANCE OF INTERACTIONS BETWEEN THE mRNA DECAY PROTEINS. III. TO EXAMINE THE NATURE AND SIGNIFICANCE OF INTERACTIONS BETWEEN THE LSM COMPLEX AND mRNA. IV. TO EXAMINE IF THE LSM PROTEINS FUNCTION IN TRANSLATION.