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

短RNA作为细胞功能的调节因子，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，以及对抗病毒RNA 沉默反应RNA沉默是指小干扰RNA（siRNA）介导的后干扰。 转录基因调控，导致病毒基因和转基因的沉默。这样的主人- 病毒相互作用涉及高度特异性、适应性、移动的和系统性的过程， 本质上是一种基于RNA的免疫反应。siRNA由19至23个碱基对的双链体组成， 具有2-nt S '-突出端和5'-磷酸，作为RNA诱导的沉默复合物（RISC）的一部分， 靶向互补病毒mRNA并标记它们以用于降解。病毒进化沉默 抑制蛋白通过修饰积累和/或活性来抵消RNA沉默 与抗病毒反应相关的siRNA。本申请建议继续我们的 正在进行的与RISC介导的RNA相关的蛋白质-RNA复合物的结构-功能研究 沉默，以及通过进化的病毒抑制蛋白的沉默抑制。我们集团 已经解析了人PAZ-siRNA和A.闪电Piwi-siRNA复合物， 曲霉aeolicus Argonaute（Ago）在游离和外部siRNA结合状态下，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 提供一个框架，用于定义额外的实验，以破译功能状态， 细菌和人Ago在结合、加工和释放过程中的构象转变 与RISC介导的催化循环相关的指导RNA和mRNA。我们期望 与托马斯合作进行的结构-突变-功能研究 洛克菲勒大学的Tuschl实验室将提供无与伦比的机械见解 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- siRNA结合的p19病毒抑制因子上的RNA识别正在扩展到p21 抑制器，其采用八聚环架构。这些研究最终将提供一个 为开发实现发育阶段或组织特异性 表达病毒抑制子，从而抑制植物中的细胞特异性沉默途径， 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.