Modulation of internal ribosome entry by ribosomal protein RPS25

描述(申请人提供)：由RNA病毒感染，其mRNAs由内部核糖体进入(IRES)机制翻译，如小核糖核酸病毒和丙型肝炎病毒(丙型肝炎病毒)，仍然是一个重大的健康威胁。例如，鼻病毒会导致普通感冒和哮喘加重，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，但不存在针对小核糖核酸病毒的有效抗病毒试剂。对于丙型肝炎病毒，有几种新的化合物正在上市，它们的疗效正在监测中。我们一直在想，这些病毒基因组中的IRES元件是否是病毒的致命弱点，我们一直在寻找专门的核糖体群体是否可能参与内部启动。我们发现，在感染脊髓灰质炎病毒过程中被修饰的核糖体蛋白RPS25，对于含有IRES的病毒RNA的翻译是必不可少的，奇怪的是，对于登革病毒也是如此，登革热病毒的mRNA是以帽依赖的方式翻译的。在这一应用中，我们建议使用各种基于细胞的分析和大量的单分子方法来研究RPS25在IRES介导的翻译中的作用，这些方法理想地应用于异质系统，如修饰的核糖体。第一个具体目标是使用一个缺乏RPS25的可行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，在核糖体图谱实验中确定。新的交联法将被用来定位活细胞中的mRNA-RPS25相互作用。目的2建议利用翻译能力提取和各种信使核糖核酸靶标研究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，利用最先进的单分子方法研究IRES介导的翻译的动力学和动力学以及RPS25变体的作用。最后一个目的是考察RPS25中特定的修改对IRES介导型翻译的影响。总体而言，这项申请将解决不同mRNAs翻译中“专门化核糖体”的基本方面。这些研究的结果可能详细说明核糖体在真核细胞中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地点。

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.