siRNA-Mediated Silencing of Viral Infection & Viral Suppressors of RNA Silencing

短RNA作为细胞功能的调节器，可以影响基因组完整性的维持 和稳定性，对细胞生长、分化和发育过程以及抗病毒RNA的影响 静音响应。RNA沉默是指小干扰RNA(SiRNA)介导的后 转录基因调控，导致病毒基因和转基因沉默。这样的东道主- 病毒相互作用涉及高度特异性、适应性、流动性和系统性的过程，这些过程在 本质上是一种基于RNA的免疫反应。SiRNA，由19到23个碱基对双链组成， 用2-NT S‘-悬垂和5’-磷酸作为RNA诱导沉默复合体的一部分， 靶向免费的病毒mRNAs并标记它们的降解。病毒进化为沉默 抑制蛋白通过改变RNA的积累和/或活性来对抗RNA沉默 与抗病毒反应相关的siRNA。此应用程序建议继续我们的 RISC介导的RNA相关蛋白质-RNA复合体的结构与功能研究进展 沉默，以及通过进化的病毒抑制蛋白抑制沉默。我们的团队已经 已经解决了人Paz-siRNA和黄曲霉Piwi-siRNA复合体的晶体结构， 在游离态和外源siRNA结合态，以及病毒 抑制子p19和p21分别处于siRNA结合和游离状态。这些结构 提供定义额外实验的框架，以破译功能状态和 细菌和人AGO在结合、加工和释放过程中的构象变化 引导RNA和与RISC介导的催化循环相关的mRNA。我们的期望是 与托马斯研究所合作进行的拟议的结构-突变-功能研究 洛克菲勒大学的Tuschl实验室将提供对机械学的无与伦比的见解 与RNA沉默途径的各个步骤相关的问题。我们对蛋白质的初步研究- 对siRNA结合的p19病毒抑制子的RNA识别正在扩展到p21 抑制器，采用八聚体环状结构。这样的研究最终应该会提供一种 为开发实现发育阶段或特定于组织的方法奠定坚实基础 病毒抑制因子的表达，从而抑制植物和 动物。

Short RNAs, as regulators of cellular function, can impact on the maintenance of genomic integrity and stability, on cell growth, differentiation and developmental processes, and on the antiviral RNA silencing response. RNA silencing refers to small interfering RNA (siRNA)-mediated post- transcriptional gene regulation, resulting in the silencing of viral genes and transgenes. Such host- virus interactions involve highly specific, adaptive, mobile and systemic processes that operate in essence as a RNA-based immune response. siRNAs, made up of 19 to 23 base pair duplexes, with 2-nt S'-overhangs and 5'-phosphates, as part of the RNA-induced silencing complex (RISC), target complimentary viral mRNAs and tag them for degradation. Viruses evolve silencing suppressor proteins to counteract RNA silencing by modifying the accumulation and/or the activity of siRNAs associated with the anti-viral response. This application proposes to continue our ongoing structure-function studies of protein-RNA complexes associated with RISC-mediated RNA silencing, as well as silencing suppression by evolved viral suppressor proteins. Our group has already solved the crystal structures of human PAZ-siRNA and A. fulgidus Piwi-siRNA complexes, of A. aeolicus Argonaute (Ago) in the free and externally siRNA-bound states, as well as the viral suppressors p19 and p21 in the siRNA-bound and free states, respectively. These structures provide a framework for defining additional experiments to decipher the functional states and conformational transitions of bacterial and human Ago's during binding, processing and release of guide RNA and mRNA associated with the RISC-mediated catalytic cycle. It is our expectation that the proposed structural-mutational-functional studies undertaken in collaboration with the Thomas Tuschl laboratory at Rockefeller University will provide unparalleled insights into mechanistic issues associated with individual steps of the RNA silencing pathway. Our initial studies of protein- RNA recognition on the siRNA-binding p19 viral suppressor are being extended to the p21 suppressor, which adopts an octameric ring architecture. Such studies should eventually provide a solid foundation for developing approaches that achieve developmental-stage or tissue-specific expression of viral suppressors, thereby inhibiting cell-specific silencing pathways in plants and animals.