Modulation of internal ribosome entry by ribosomal protein RPS25

核糖体蛋白 RPS25 对内部核糖体进入的调节

• 批准号: 9412429
• 负责人: JOSEPH D PUGLISI
• 金额: $ 58.03万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9412429
• 关键词: Address; Affect; Affinity; Antiviral Agents; Antiviral Therapy; Asia; Asthma; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Cell Line; Cells; Common Cold; Complex; Dengue; Dengue Vaccine; Dengue Virus; Ectopic Expression; Elements; Enterovirus 71; Epidemic; Eukaryotic Cell; Family Picornaviridae; Fluorescence; Gene Expression; Genes; Goals; Growth; Haploid Cells; Health; Hepatitis A Virus; Hepatitis C virus; Human poliovirus; Impairment; Infection; Internal Ribosome Entry Site; Kinetics; Knock-out; Label; Ligands; Maps; Mass Spectrum Analysis; Measures; Mediating; Messenger RNA; Modification; Molecular Conformation; Monitor; Mutate; Mutation; Nanostructures; Outcome Study; Picornaviridae Infections; Poliovirus Vaccines; Population; Process; Production; Proto-Oncogenes; RNA Virus Infections; RNA Viruses; Reagent; Rhinovirus; Ribosomal Interaction; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Series; System; Time; Translating; Translation Initiation; Translations; Universities; Untranslated RNA; Variant; Viral; Viral Genome; Viral Proteins; Virus; base; cancer therapy; crosslink; experimental study; healing; laser tweezer; novel; photonics; polysome profiling; public health relevance; recruit; ribosome profiling; single molecule; viral RNA

DESCRIPTION (provided by applicant): Infection by RNA viruses, whose mRNAs are translated by an internal ribosome entry (IRES) mechanism, such as picornaviruses and hepatitis C virus (HCV), remains a significant health threat. For example, rhinovirus causes the common cold and exacerbation of asthma, and enterovirus 71 is currently epidemic in parts of Asia. Notwithstanding the poliovirus vaccines, no effective antiviral reagent exists for picornaviruses. For HCV, several new compounds are being on the market and their efficacies are being monitored. We have been wondering whether the IRES elements in these viral genomes present an Achilles' heel for the viruses, and we have been searching whether specialized ribosome populations might be involved in internal initiation. We discovered that ribosomal protein RPS25, which is modified during infection with poliovirus, is essential for translation of IRES-containing viral RNAs and curiously, also for Dengue virus who's mRNA is translated in a cap-dependent manner. In this application, we propose to study roles for RPS25 in IRES-mediated translation, using a variety of cell-based assays and a large array of single-molecule approaches that are ideally applied to heterogeneous systems such as modified ribosomes. The first specific aim proposes to use a viable haploid cell that lacks RPS25 to study interactions of ribosomes with known IRES-containing RNAs, Dengue viral RNAs and cellular RNAs, that require RPS25, identified in ribosomal profiling experiments. Novel crosslinking assays will be used to map mRNA-RPS25 interactions in living cells. Aim 2 proposes to study steps in translation that are affected by RPS25 using translation competent-extract and a variety of mRNA targets. Specific Aim 3 details a very comprehensive approach to study the dynamics and kinetics of IRES-mediated translation and the role for RPS25 variants, employing state-of-the-art single molecule approaches. The last aim proposes to examine effects of specific identified modifications in RPS25 on IRES-mediated translation. Overall, this application will address fundamental aspects of "specialized ribosomes" in the translation of distinct mRNAs. The outcomes of these studies are likely to detail novel mechanisms of gene expression mediated by the ribosome in eukaryotic cells and may point to new venues for antiviral and anticancer therapies.

描述（由申请人提供）：RNA 病毒（其 mRNA 通过内部核糖体进入 (IRES) 机制翻译）感染，例如小核糖核酸病毒和丙型肝炎病毒 (HCV)，仍然是重大的健康威胁。例如，鼻病毒会导致普通感冒和哮喘恶化，而肠道病毒71型目前在亚洲部分地区流行。尽管有脊髓灰质炎病毒疫苗，但尚不存在针对小核糖核酸病毒的有效抗病毒试剂。对于 HCV，几种新化合物正在上市，其功效正在接受监测。我们一直想知道这些病毒基因组中的 IRES 元件是否是病毒的致命弱点，并且我们一直在寻找特殊的核糖体群体是否可能参与内部启动。我们发现核糖体蛋白 RPS25 在脊髓灰质炎病毒感染过程中被修饰，对于含有 IRES 的病毒 RNA 的翻译至关重要，奇怪的是，对于登革热病毒来说也是如此，因为登革热病毒的 mRNA 以帽子依赖性方式翻译。在此应用中，我们建议使用各种基于细胞的测定和大量单分子方法来研究 RPS25 在 IRES 介导的翻译中的作用，这些方法非常适合应用于异质系统（例如修饰核糖体）。第一个具体目标建议使用缺乏 RPS25 的活单倍体细胞来研究核糖体与已知的含有 IRES 的 RNA、登革热病毒 RNA 和细胞 RNA 的相互作用，这些 RNA 需要 RPS25，在核糖体分析实验中鉴定出。新型交联测定将用于绘制活细胞中 mRNA-RPS25 相互作用的图谱。目标 2 提议使用翻译活性提取物和各种 mRNA 靶标来研究受 RPS25 影响的翻译步骤。具体目标 3 详细介绍了一种非常全面的方法，采用最先进的单分子方法来研究 IRES 介导的翻译的动力学和动力学以及 RPS25 变体的作用。最后一个目标是检查 RPS25 中特定修饰对 IRES 介导的翻译的影响。总体而言，该应用将解决不同 mRNA 翻译中“特殊核糖体”的基本问题。这些研究的结果可能会详细说明真核细胞中核糖体介导的基因表达的新机制，并可能为抗病毒和抗癌治疗指明新的途径。