Modulation of internal ribosome entry by ribosomal protein RPS25

描述（由申请人提供）：RNA病毒感染，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），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加重，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，但没有有效的小核糖核酸病毒抗病毒试剂。对于丙型肝炎病毒，几种新的化合物正在上市，它们的功效正在受到监测。我们一直想知道这些病毒基因组中的IRES元素是否存在病毒的致命弱点，并且我们一直在寻找特定的核糖体群体是否可能参与内部起始。我们发现核糖体蛋白RPS25在脊髓灰质炎病毒感染期间被修饰，对于翻译含ires的病毒rna至关重要，奇怪的是，对于以帽依赖方式翻译mRNA的登革热病毒也是如此。在这个应用中，我们建议研究RPS25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，如修饰核糖体。第一个具体目标是使用缺乏RPS25的活的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和需要RPS25的细胞rna的相互作用，这些rna是在核糖体分析实验中发现的。新的交联分析将用于绘制活细胞中mRNA-RPS25的相互作用。目的2提出利用翻译能力提取物和多种mRNA靶点研究受RPS25影响的翻译步骤。具体目标3详细介绍了一个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RPS25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RPS25中特定的修饰对ires介导的翻译的影响。总的来说，这一应用将解决不同mrna翻译中“特化核糖体”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。

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.