THE ROLE OF THE RIBOSOME IN DETERMINING THE FATE OF DAMAGED MRNA

 描述(申请人提供)：快速和忠实地翻译细胞信使RNA是核糖体和翻译因素的一个定义特征。高精度的蛋白质合成确保了更容易错误折叠的错误蛋白质不会被制造出来。对于异常的RNA，如截短的和含有过早或缺少终止密码子的RNA，一些基于核糖体的质量控制过程确保这些RNA不被翻译，而是以降解为目标。虽然在过去的十年里，这些信使核糖核酸监测机制受到了很大的关注，但奇怪的是，另一类异常的信使核糖核酸却几乎没有得到研究。特别是，化学损伤的RNA对翻译的保真度和效率构成了一个重大障碍。对这个提议特别感兴趣的是氧化和烷基化的信使核糖核酸。有趣的是，我们最近的数据表明，氧化的mRNAs阻碍了翻译，似乎利用了已经描述的mRNAs监控过程。我的实验室的长期目标是扩大我们对质量控制过程的理解，这些过程负责识别受损的RNA及其对细胞健康的影响。在这一建议中，我们主张核糖体在识别过程在核糖体的解码中心启动的途径中发挥积极作用。在tRNA选择过程中，解码的变化可能会触发一个信号级联反应，利用现有的质量控制过程来针对降解的mRNA。近期目标是通过使用定义明确的高分辨率体外系统来研究氧化和烷基化碱基对核糖体解码的影响(目标1)。这一目标是在突变平移因素的背景下，围绕一系列稳定前动力学方法而建立的。通过建立核糖体对受损mRNAs反应的动力学和热力学框架，我们希望不仅能确定受损RNA对细胞代谢造成不良后果的分子机制，而且还能确定可能对下游质量控制过程负责的信号信号。为此，我们还计划调查这些RNA随后如何被细菌和真核生物降解，以及核糖体是否在这一过程中发挥积极作用(目标2)。我们建议使用无偏见的方法，这将使我们能够在全球范围内评估细胞中RNA损伤的图景，以及核糖体和信使核糖核酸监控因素如何改变这一图景。我们还计划引入在特定位置受损的mRNAs，以克服研究受损mRNAs命运的一些困难。最后，尽管细胞对DNA损伤的反应一直是许多研究的主题，但对RNA损伤的反应很少或根本没有受到关注。我们有初步数据表明，细菌的适应性反应是由RNA损伤触发的。在目标3中，我们建议对这种活动进行生物物理学表征，并研究其对细胞反应的作用。总体而言，这项建议中描述的实验解决了关键的生物学问题，这些问题可能会对我们理解细胞对破坏性物质的反应产生广泛影响。

 DESCRIPTION (provided by applicant): Fast and faithful translation of the cellular messenger RNAs is a defining feature of the ribosome and the translation factors. High-accuracy protein synthesis ensures that errant proteins, which are more prone to misfold, are not made. On aberrant mRNAs, such as truncated ones and those either containing premature or lacking stop codons, a number of ribosome-based quality control processes ensure that these RNAs are not translated and instead are targeted for degradation. While these mRNA-surveillance mechanisms have received much attention during the past decade, curiously a different class of aberrant mRNAs has received little study. In particular, chemically-damaged RNAs pose a significant hurdle to translational fidelity and efficiency. Of particular interest to this proposa are oxidized and alkylated mRNA. Interestingly, our recent data suggests that oxidized mRNAs stall translation and appear to utilize already described mRNA-surveillance processes. The long-term goal of my laboratory is to expand our understanding of the quality control processes that are responsible for recognizing damaged RNAs and their impact on cellular fitness. In this proposal, we argue for an active role for the ribosome in the pathway in which the recognition process initiates in the decoding center of the ribosome. Changes in decoding during tRNA selection are likely to trigger a signaling cascade taking advantage of existent quality control processes to target the mRNA for degradation. The immediate goal is to study the effect of oxidized and alkylated bases on decoding by the ribosome through the use of a well-defined high-resolution in vitro system (aim 1). This goal is built around a range of pre-steady- state kinetics approaches in the context of mutated translation factors. By establishing a kinetic and thermodynamic framework for the ribosomal response to damaged mRNAs, we hope not only to define the molecular mechanism of the unwanted consequence of damaged RNA on cellular metabolism but also define signaling cues that are likely responsible for downstream quality control processes. To this end, we also plan to investigate how these RNAs are subsequently targeted for degradation in bacteria and eukaryotes and whether the ribosome plays an active role in the process (aim 2). We propose to use unbiased approaches that will allow us to globally assess the landscape of RNA damage in the cell and how the ribosome and mRNA- surveillance factors alter this landscape. We also plan to introduce mRNAs damaged at specific sites to overcome some of the difficulties of studying the fate of damaged mRNAs. Finally, whereas the cellular response to DNA damage has been the subject of many studies, the response to RNA damage has received little to no attention. We have preliminary data that suggests that the bacterial adaptive response is triggered by RNA damage. In aim 3 we propose to biophysically characterize this activity and study its utility to cellular response. Overall, th experiments described in this proposal address key biological problems that are likely to have a broad impact on our understanding of the cellular response to damaging agents.