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

核糖体在决定受损 mRNA 命运中的作用

• 批准号: 9115638
• 负责人: Hani Zaher
• 金额: $ 30.12万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-27 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9115638
• 关键词: 8-hydroxyguanosine; Active Sites; Address; Adenosine; Affect; Alkylation; Amino Acyl Transfer RNA; Antibiotics; Attention; Bacteria; Base Pairing; Biological; Cells; Codon Nucleotides; Cues; Cultured Cells; DNA Damage; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Disease; Ensure; Enzymes; Escherichia coli; Eukaryota; Gene Expression; Goals; Health; High-Throughput Nucleotide Sequencing; In Vitro; Kinetics; Laboratories; Lesion; MGMT gene; Measures; Messenger RNA; Metabolism; Molecular; Mutate; Nature; Neurodegenerative Disorders; Nucleic Acids; Nucleotides; Open Reading Frames; Pathogenicity; Pathway interactions; Play; Positioning Attribute; Procedures; Process; Property; Protein Biosynthesis; Proteins; Quality Control; RNA; Reporter; Resolution; Ribosomes; Role; Signal Transduction; Singlet Oxygen; Site; Speed; Stress; Suicide; System; Techniques; Terminator Codon; Testing; Thermodynamics; Transfer RNA; Transferase; Translating; Translations; Untranslated Regions; activating transcription factor; adduct; assault; base; chemotherapy; coping; fitness; in vivo; inhibitor/antagonist; interest; knock-down; mRNA Surveillance; mRNA Transcript Degradation; methyl group; mutant; oxidation; oxidative damage; premature; programs; repaired; research study; response; sensor; stopped-flow fluorescence; success; translation factor

 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.

 描述（适用提供）：蜂窝信使RNA的快速而忠实的翻译是核糖体和翻译因子的定义特征。高准确的蛋白质合成可确保不会产生更容易折叠的误差蛋白。在异常的mRNA（例如截短的mRNA和包含过早的密码子的mRNA）上，许多基于核糖体的质量控制过程确保没有翻译这些RNA，而是针对降解的目标。尽管在过去的十年中，这些mRNA检查机制引起了很多关注，但老实说，不同类别的异常mRNA几乎没有得到研究。特别是，化学损害的RNA在翻译的忠诚度和效率上构成了重大障碍。该提案特别感兴趣的是氧化和烷基化的mRNA。有趣的是，我们的最新数据表明，氧化的mRNA档位翻译，似乎利用已经描述了mRNA保留过程。我实验室的长期目标是扩大我们对负责识别RNA损坏及其对细胞适应性的影响的质量控​​制过程的理解。在此提案中，我们主张核糖体在核糖体解码中心启动识别过程中的核糖体作用。 tRNA选择过程中解码的变化可能会触发信号传导级联，利用现有的质量控制过程来靶向mRNA以降解。直接的目标是研究氧化和烷基化碱的作用对核糖体通过使用明确定义的高分辨率体外系统解码的作用（AIM 1）。该目标围绕着突变的翻译因素的背景下围绕一系列稳态的态动力学方法构建。通过建立一个对受损的mRNA的核糖体反应的动力学和热力学框架，我们不仅希望定义RNA在细胞代谢中受损不良后果的分子机制，而且还定义了可能导致下游质量控制过程的信号线索。为此，我们还计划研究这些RNA如何针对细菌和真核生物中的降解以及核糖体是否在此过程中起积极作用（AIM 2）。我们建议采用公正的方法，使我们能够在全球范围内评估细胞中RNA损伤的景观以及核糖体和mRNA-监测因子如何改变这种景观。我们还计划在特定地点引入损坏的mRNA，以克服研究受损的mRNA脂肪的一些困难。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损伤的反应几乎没有引起注意。我们有初步数据表明细菌自适应反应是由RNA损伤触发的。在AIM 3中，我们建议生物物理表征该活性并研究其对细胞反应的效用。总体而言，该提案中描述的实验解决了关键的生物学问题，这些问题可能会对我们对损伤剂的细胞反应的理解产生广泛影响。