Modulation of internal ribosome entry by ribosomal protein RPS25

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）（如小核糖核酸病毒和丙型肝炎病毒（HCV））的感染仍然是一个重大的健康威胁。例如，鼻病毒引起普通感冒和哮喘恶化，肠道病毒71目前在亚洲部分地区流行。尽管有脊髓灰质炎病毒疫苗，但没有针对小核糖核酸病毒的有效抗病毒试剂。对于HCV，几种新的化合物正在上市，其疗效正在监测中。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，我们一直在寻找是否专门的核糖体群体可能参与内部启动。我们发现核糖体蛋白RPS 25在脊髓灰质炎病毒感染期间被修饰，对于含有IRES的病毒RNA的翻译是必需的，并且奇怪的是，对于登革病毒的mRNA以帽依赖性方式翻译也是必需的。在这个应用中，我们建议研究RPS 25在IRES介导的翻译中的作用，使用各种基于细胞的测定和大量的单分子方法，这些方法理想地应用于异质系统，如修饰的核糖体。第一个具体目标提出使用缺乏RPS 25的活的单倍体细胞来研究核糖体与已知的含IRES的RNA、登革热病毒RNA和细胞RNA的相互作用，所述RNA需要RPS 25，在核糖体分析实验中鉴定。新的交联测定将用于绘制活细胞中mRNA-RPS 25相互作用。目的2提出使用翻译能力提取物和各种mRNA靶标研究RPS 25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来研究IRES介导的翻译的动力学和动力学以及RPS 25变体的作用，采用最先进的单分子方法。最后一个目的是研究RPS 25中特定修饰对IRES介导的翻译的影响。总的来说，本申请将解决在不同mRNA的翻译中的“特化核糖体”的基本方面。这些研究的结果可能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。

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.