mRNA Decapping in Yeast

描述(申请人提供)：我们工作的长期目标是了解控制真核细胞信使核糖核酸稳定性的分子机制，这是基因调控的关键步骤。真核生物信使核糖核酸转换的一个主要途径是多聚(A)尾巴缩短，它触发解帽，导致5‘-3’外切核解。剪接是这一途径中的关键步骤，因为它诱导了mRNA的降解，并且是许多控制输入的位置，包括Poly(A)尾巴和调节mRNA解压速率的特定序列。鉴于这一重要性，我们专注于了解mRNA解离的机制。在过去的资金支持期间，我们澄清了解帽解酶的性质，确定了一些蛋白质影响解帽速率的机制，并提供了强有力的证据表明，解帽的关键步骤是退出mRNAs翻译池，并结合特定的mRNP重排。此外，我们还发现了新的细胞质结构(称为P-小体)，在这些结构中，mRNA的分解机制集中在那里，并且可以发生mRNA的降解。P小体具有重要意义，因为：1)它们暗示了未预料到的信使核糖核酸衰变的区区化，这可能成为信使核糖核酸调控的一个点；2)在其他真核生物中是保守的；3)可能是信使核糖核酸生物学中额外事件的发生部位，可能包括翻译抑制的信使核糖核酸的存储。在这项资助中，我们将继续我们的分析，重点是了解P小体的组成、性质和功能。具体目标如下： I)测定P小体的组成和超微结构。 Ii)分析P形体总成。 Iii)分析Dhh1和Pat1蛋白的功能。 IV)确定P-小体是否在细胞质mRNA生理的其他方面起作用。

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.