mRNA Decapping in Yeast

我们工作的长期目标是了解控制真核生物mRNA的分子机制 稳定性，这是基因调控的关键步骤。真核生物中mRNA周转的一个主要途径 起始于poly（A）尾缩短，其触发去帽，导致5'至3'外切核溶解。去盖 是这一途径中的关键步骤，因为它诱导mRNA的降解，并且是许多 控制输入，包括poly（A）尾和调节mRNA去帽速率的特定序列。给定 由于这种重要性，我们专注于理解mRNA去帽的机制。在过去的融资 在此期间，我们阐明了脱帽酶的性质，确定了一些蛋白质 影响去盖的速度，并提供了强有力的证据表明，去盖的一个关键步骤是退出 与特定mRNP重排偶联的mRNA翻译池。此外，我们还发现， 新的细胞质结构（称为P体），其中mRNA去帽机制是 浓缩的，并且mRNA降解可以发生的地方。P体是重要的，因为：1）它们意味着 mRNA衰变的意外区室化，这成为mRNA调节的可能点， 2)在其他真核生物中是保守的，3）可能是mRNA生物学中额外事件的位点， 可能包括储存抑制的mRNA。在这份研究报告中，我们将继续分析 mRNA去帽，重点是了解P体的组成，性质和功能。的 具体目标如下： I）确定P体的组成和超微结构 II）分析P体装配 III）分析Dhhl和Pat1蛋白的功能 IV）确定P体是否在细胞质mRNA生理学的其他方面起作用。

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 Dhhl and Pat1 proteins IV) Determine if P-bodies function in other aspects of cytoplasmic mRNA physiology.