Investigating the process of exiting mRNAs out of translation

描述(申请人提供)：本方案的长期目标是详细了解停止信使核糖核酸翻译的过程。我们发现了令人信服的证据表明，关闭mRNA翻译不仅仅是一个简单的被动和默认事件，而是mRNA被主动从翻译机制中移除，以响应特定的提示。重要的是，我们已经确定依赖于ATP的RNA解旋酶Dhh1p是mRNA从翻译中解离所必需的因子。此外，数据表明，Dhlp在mRNA的3‘端失去Poly(A)尾巴后，具有退出翻译的功能。我们还证明了在营养饥饿的条件下，Dhlp被细胞用来实现对mRNA表达的全局调控。因此，Dhlp既是顺式活性的，也是顺式活性的。正常细胞生长条件下的信使核糖核酸翻译调节因子和细胞应激时信使核糖核酸表达的全局调节因子。我们建议在三个特定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Dhlp在抑制营养饥饿诱导的全球mRNA翻译中的作用。我们的初步观察表明，在葡萄糖和氨基酸饥饿的情况下，mRNA3‘聚(A)尾的状态或功能会迅速和彻底地改变。我们将研究Poly(A)尾巴功能的这种戏剧性变化是如何实现的，以及这是否是应激时Dhlp将mRNA移动到翻译静止所必需的。其次，我们将剖析所观察到的Dhlp与翻译过程中的mRNA之间相互作用的性质。特别是，我们将确定Dhlp与翻译的mRNAs相互作用的方式，以及Dhlp是否与核糖体或与mRNAs本身特异性相互作用。最后，我们将研究在正常和营养胁迫条件下，Dhlp促进mRNA退出翻译的分子机制。初步证据表明，Dhlp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Dhlp的其他底物。综上所述，我们将进一步探索依赖于Dhlp的mRNA从翻译中移除的机制。重要的是，Dhlp的活性似乎在功能上是保守的。在卵母细胞中，Dhlp的同源基因对于翻译沉默的母体mRNAs是至关重要的，突变导致翻译沉默的母体mRNA性早熟激活和胚胎缺陷的表现。与这些观察结果一致的是，小鼠和人类Dhlp通过影响mRNA翻译参与配子发生和癌症发生。因此，拟议的研究可以为公共卫生问题提供相关的见解，如出生缺陷、不孕不育和细胞转化的表现。

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.