Role of Argonaute in miRNA biogenesis and function

描述（由申请人提供）：在过去的十年中，调控rna控制生物途径的发现彻底改变了我们对基因表达的理解。在最前沿，microRNAs （miRNAs）已被证明是植物和动物中丰富而重要的一类RNA分子。新的认识强调了miRNA在人类生物学中的重要性，即特定miRNA通路的错误调节是复杂疾病的基础，包括癌症、心脏病和神经病理学。为了理解miRNA在正常和疾病条件下的作用，必须解决两个基本问题：(1)miRNA的表达是如何调控的，以及在疾病状态下miRNA的表达是如何改变的；(2)生物学上相关的miRNA靶点是什么，其调控不当导致疾病表型？这些问题是本文提出的工作的基础，旨在阐明控制miRNA生物发生的新机制，并确定miRNA复合物如何识别和调节体内特定靶标。通常，mirna将AGO及其辅助因子招募到特定的mrna中，以触发衰变或翻译抑制。在这里，AGO在调节miRNA生物发生中的全新作用将被研究。在Aim 1中，AGO用于增强let-7和其他mirna加工的机制将在秀丽隐杆线虫和人类细胞中确定。这些研究将揭示一种控制miRNA表达的新途径，这可能与理解miRNA水平在发育和疾病进展过程中如何波动广泛相关。Aim 2将解决一个令人生畏的问题，即不完美的碱基配对如何足以满足mirna对特定靶标的调节。将采用三种互补的方法来解读AGO如何识别和调节内源性环境中的特定目标。这些策略利用敏感的生化方法，独特的蠕虫菌株和强大的计算管道来评估AGO在秀丽隐杆线虫中由特定mirna引导的全基因组靶向特性。AGO与其靶标结合的后果也将被分析，以提供miRNA在活体动物中的功能的全面视图。最终，这些研究将产生前所未有的数据集，用于破译多细胞动物体内miRNA靶标识别和调控的规则。

DESCRIPTION (provided by applicant): The discovery that regulatory RNAs control biological pathways have revolutionized our understanding of gene expression over the past decade. At the forefront, microRNAs (miRNAs) have proven to be an abundant and essential class of RNA molecules in plants and animals. The importance of miRNAs in human biology is highlighted by the new recognition that mis-regulation of specific miRNA pathways underlies complex diseases, including cancer, heart ailments and neuronal pathologies. To understand the role of miRNAs under normal and disease conditions, two basic questions must be addressed: (1) how is miRNA expression regulated and how is this altered in the disease state, and (2) what are the biologically relevant miRNA targets, whose mis-regulation causes the disease phenotypes? These problems are the foundation of the work proposed here to elucidate a novel mechanism for controlling miRNA biogenesis and to determine how the miRNA complex recognizes and regulates specific targets in vivo. Typically, miRNAs recruit Argonaute (AGO) and its co-factors to specific mRNAs to trigger decay or translational repression. Here, an entirely new role for AGO in regulating miRNA biogenesis will be investigated. In Aim 1, the mechanism used by AGO to enhance the processing of let-7, and potentially other miRNAs, will be determined in C. elegans and human cells. These studies will reveal a novel pathway for controlling miRNA expression that may be broadly relevant for understanding how miRNA levels fluctuate during development and disease progression. Aim 2 will tackle the daunting problem of how imperfect base-pairing suffices for the regulation of specific targets by miRNAs. Three complementary methods will be employed to decipher how AGO recognizes and regulates specific targets in the endogenous context. These strategies take advantage of sensitive biochemical methods, unique worm strains and robust computational pipelines to assess the genome wide targeting properties of AGO guided by specific miRNAs in C. elegans. The consequences of AGO binding to its targets will also be analyzed to provide a comprehensive view of miRNA function in a live animal. Ultimately, these studies will yield unprecedented datasets for deciphering the rules used in vivo for miRNA target recognition and regulation in a multicellular animal.