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

描述(申请人提供)：短RNA作为细胞功能的调节器，可以影响基因组完整性和稳定性的维持，对细胞的生长、分化和发育过程，以及对抗病毒RNA沉默反应的影响。RNA沉默是指小干扰RNA(SiRNA)介导的转录后基因调控，导致病毒基因和转基因沉默。这种宿主与病毒的相互作用涉及高度特异性、适应性、移动性和系统性的过程，本质上是作为基于RNA的免疫反应运行的。作为RNA诱导沉默复合体(RISC)的一部分，siRNAs由19到23个碱基对双链组成，带有2-NT，3‘-突出端和5’-磷酸，针对互补的病毒mRNAs并标记它们进行降解。病毒通过改变与抗病毒反应相关的siRNAs的积累和/或活性来进化沉默抑制蛋白来抵消RNA沉默。这项申请建议继续我们正在进行的与RISC介导的RNA沉默相关的蛋白质-RNA复合体的结构-功能研究，以及进化的病毒抑制蛋白对沉默的抑制。本课题组已经解决了人Paz-siRNA和A.fulgidus Piwi-siRNA复合体的晶体结构，以及病毒抑制基因p19和p21分别处于siRNA结合和游离状态。这些结构提供了一个框架，用于定义额外的实验，以破译细菌和人类AGO在结合、处理和释放与RISC介导的催化循环相关的引导RNA和mRNA过程中的功能状态和构象转变。我们期望，与洛克菲勒大学Thomas Tuschl实验室合作进行的拟议结构突变功能研究将为与RNA沉默途径的个别步骤相关的机制问题提供无与伦比的见解。我们对siRNA结合的p19病毒抑制子上蛋白质-RNA识别的初步研究正在扩展到采用八聚体环结构的p21抑制子。这些研究最终应该为开发实现病毒抑制物在发育阶段或组织特异性表达的方法提供坚实的基础，从而抑制植物和动物中的细胞特异性沉默途径。

DESCRIPTION (provided by applicant): 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 3'-overhangs and 5'-phosphates, as part of the RNA-induced silencing complex (RISC), target complementary 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.