Investigating the process of exiting mRNAs out of translation

描述（由申请人提供）：本提案的长期目标是详细了解mRNA翻译停止的过程。我们已经发现了令人信服的证据，表明关闭mRNA翻译不仅仅是mRNA生命周期中的被动和默认事件，而是mRNA在响应特定提示时主动从翻译机制中移除。重要的是，我们已经确定了atp依赖的RNA解旋酶Dhh1 p，作为mRNA从翻译中解离所需的因子。此外，数据表明Dhhlp的功能是在mRNA 3'端poly(A)尾巴缺失后退出mRNA的翻译。我们还证明，在营养匮乏的条件下，Dhhlp被细胞用于实现mRNA表达的全局调控。因此，dhhelp的行为既是顺式行为。正常细胞生长条件下mRNA翻译的调节因子和细胞应激下mRNA表达的全局调节因子。我们建议在三个具体目标下扩展我们对这一未探索的基因调控步骤的初步鉴定。首先，我们将研究dhhelp在营养饥饿诱导的全球mRNA翻译抑制中的作用。我们的初步观察表明，在葡萄糖和氨基酸饥饿的情况下，mRNA 3' poly(A)尾巴的状态或功能会迅速而彻底地改变。我们将研究poly(A)尾部功能的这种戏剧性改变是如何实现的，以及在应激条件下dhhelp是否需要mRNA进入翻译静止状态。其次，我们将剖析观察到的Dhhlp和mRNA之间相互作用的性质。特别是，我们将确定Dhhlp与翻译mRNA相互作用的模式，以及Dhhlp是否特异性地与核糖体或mRNA本身相互作用。最后，我们将探讨dhhelp在正常和营养胁迫条件下促进mRNA退出翻译的分子机制。初步证据表明dhhelp活性的底物是40S核糖体亚基本身。这种相互作用的性质将得到更严格的测试，并将进行遗传和生化分析，以确定dhhelp的其他底物。总之，我们将进一步探索依赖dhhelp的mRNA从翻译中移除的机制。重要的是，dhhelp活性似乎在功能上是保守的。dhhelp的同源物对于母源mRNA在卵母细胞中的翻译沉默至关重要，突变导致翻译沉默的母源mRNA的早熟激活和胚胎缺陷的表现。与这些观察结果一致，小鼠和人类Dhhlp通过影响mRNA翻译参与配子发生和致癌。因此，拟议的研究可以预期为诸如出生缺陷、不育和细胞转化的表现等公共卫生问题提供相关的见解。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand, in detail, the process of cessation of mRNA translation. We have uncovered compelling evidence that shutting down mRNA translation is not simply a passive and default event in the lifetime of an mRNA, but, rather, that mRNA is actively removed from the translational machinery in response to specific cues. Importantly, we have identified the ATP-dependent RNA helicase, Dhh1 p, as a factor required for the dissociation of mRNA from translation. Furthermore, data suggests that Dhhlp functions to exit an mRNA from translation following loss of the poly(A) tail from the 3' end of the mRNA. We have also demonstrated that Dhhlp is used by the cell to achieve global regulation of mRNA expression under conditions of nutrient-starvation. Dhhlp acts, therefore, both as a cis-acitng . regulator of mRNA translation under normal cell growth conditions and a global regulator of mRNA expression upon cellular stress. We propose to extend our initial identification of this unexplored step of gene regulation under three specific aims. First, we will examine the role of Dhhlp in the repression of global mRNA translation that is induced by nutrient starvation. Our preliminary observations indicate that under either glucose and amino acid starvation, mRNA 3' poly(A) tail status or function is rapidly and radically altered. We will investigate how this dramatic alteration in poly(A) tail function is achieved and if it is required for the movement of mRNA into translational quiescence by Dhhlp upon stress. Second, we will dissect the nature of the observed interaction between Dhhlp and mRNA undergoing translation. In particular, we will determine the mode by which Dhhlp interacts with translating mRNAs, and if Dhhlp specifically interacts with either ribosomes or with the mRNA itself. Lastly, we will investigate the molecular mechanisms by which Dhhlp promotes the exit of mRNA out of translation under normal and nutrient stress conditions. Preliminary evidence suggests that the substrate for Dhhlp activity is the 40S ribosomal subunit itself. The nature of this interaction will be more rigorously tested, and genetic and biochemical analyses will be preformed to identify additional substrates of Dhhlp. In aggregate, we will explore further the mechanisms by which mRNA is removed from translation dependent upon Dhhlp. Importantly, Dhhlp activity appears to be functionally conserved. Homologues of Dhhlp are critical for translational silencing of maternal mRNAs in oocytes, and mutations lead to precocious activation of translationally silenced maternal mRNA and the manifestation of embryonic defects. Consistent with these observations, mouse and human Dhhlp has been implicated in gametogenesis and carcinogenesis by impacting mRNA translation. The proposed studies, therefore, can be anticipated to provide relevant insight into public health issues such as the manifestation of birth defects, sterility, and cellular transformation.