mRNA Decapping in Yeast

描述（由申请人提供）：我们工作的长期目标是了解控制真核mRNA稳定性的分子机制，这是基因调控的关键步骤。真核生物mRNA转换的一个主要途径是从poly(A)尾巴缩短开始的，它触发脱帽，导致5‘到3’外核溶解。脱帽是该途径的关键步骤，因为它诱导mRNA的降解，并且是许多控制输入的位点，包括poly(a) tail和调节mRNA脱帽率的特定序列。鉴于这一重要性，我们专注于理解mRNA脱帽的机制。在过去的资助期内，我们澄清了脱帽酶的性质，确定了一些蛋白质影响脱帽速率的机制，并提供了强有力的证据，证明脱帽的关键步骤是退出mrna的翻译池，并伴有特定的mRNP重排。此外，我们还发现了新的细胞质结构（称为p体），其中mRNA脱帽机制集中，mRNA降解可能发生。p小体之所以重要，是因为：1)它们意味着mRNA衰变的意外区隔化，这可能成为mRNA调控的一个点；2)在其他真核生物中是保守的；3)可能是mRNA生物学中其他事件的位点，可能包括翻译抑制mRNA的储存。在本研究中，我们将继续对mRNA脱帽进行分析，重点是了解p -小体的组成、性质和功能。具体目标如下：

DESCRIPTION (provided by applicant): The long-term goal of our work is to understand the molecular mechanisms that control eukaryotic mRNA stability, which is a critical step in gene regulation. One major pathway of mRNA turnover in eukaryotes initiates with poly(A) tail shortening, which triggers decapping, leading to 5' to 3' exonucleolysis. Decapping is a key step in this pathway because it induces degradation of the mRNA, and is the site of numerous control inputs including the poly(A) tail and specific sequences that modulate mRNA decapping rate. Given this importance, we focused on understanding the mechanisms of mRNA decapping. In the past funding period, we clarified the nature of the decapping enzyme, identified the mechanisms by which some proteins affect the rate of decapping, and provided strong evidence that a key step in decapping is exiting the translating pool of mRNAs coupled with a specific mRNP re-arrangement. In addition, we also identified novel cytoplasmic structures (referred to as P-bodies) where the mRNA decapping machinery is concentrated and where mRNA degradation can occur. P-bodies are significant because: 1) they imply unanticipated compartmentalization of mRNA decay, which becomes a possible point of mRNA regulation, 2) are conserved in other eukaryotes and 3) are likely to be sites of additional events in mRNA biology, possibly including storage of translationally repressed mRNAs. In this grant, we will continue our analysis of mRNA decapping with a focus on understanding the composition, properties, and function of P-bodies. The specific aims are as follows: I) Determine the composition and ultrastructure of P-bodies. II) Analyze P-body assembly. III) Analyze the function of the Dhh1 and Pat1 proteins. IV) Determine if P-bodies function in other aspects of cytoplasmic mRNA physiology.