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

 描述（由申请人提供）：细胞信使RNA的快速和忠实翻译是核糖体和翻译因子的定义特征。高精度的蛋白质合成确保了不会产生更容易错误折叠的错误蛋白质。对于异常的mRNA，例如截短的mRNA和含有过早或缺乏终止密码子的mRNA，许多基于核糖体的质量控制过程确保这些RNA不被翻译，而是被靶向降解。虽然这些mRNA监视机制在过去十年中受到了很多关注，但奇怪的是，另一类异常mRNA却很少受到研究。特别是，化学损伤的RNA对翻译保真度和效率构成了重大障碍。对该提议特别感兴趣的是氧化和烷基化的mRNA。有趣的是，我们最近的数据表明，氧化的mRNA停止翻译，似乎利用已经描述的mRNA监视过程。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。在这个提议中，我们认为核糖体在识别过程中起着积极的作用，在核糖体的解码中心启动。tRNA选择过程中解码的变化可能会触发信号级联反应，利用现有的质量控制过程靶向mRNA进行降解。近期目标是通过使用明确定义的高分辨率体外系统研究氧化和烷基化碱基对核糖体解码的影响（目的1）。这一目标是建立在一系列的前稳态动力学方法的背景下，突变的翻译因子。通过建立一个动力学和热力学框架的核糖体响应受损的mRNA，我们希望不仅要确定受损的RNA对细胞代谢的不良后果的分子机制，但也定义可能负责下游质量控制过程的信号转导线索。为此，我们还计划研究这些RNA随后如何在细菌和真核生物中被靶向降解，以及核糖体是否在该过程中发挥积极作用（目的2）。我们建议使用无偏的方法，这将使我们能够全面评估细胞中RNA损伤的景观，以及核糖体和mRNA监视因子如何改变这一景观。我们还计划引入在特定位点受损的mRNA，以克服研究受损mRNA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损伤的反应却很少受到关注。我们有初步的数据表明，细菌的适应性反应是由RNA损伤引发的。在目标3中，我们建议从生物药理学上表征这种活性，并研究其对细胞反应的效用。总的来说，本提案中描述的实验解决了关键的生物学问题，这些问题可能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.