Role of Argonaute in miRNA biogenesis and function

描述(申请人提供)：在过去的十年里，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和必要的一类RNA分子。新的认识突显了miRNAs在人类生物学中的重要性，即特定miRNA途径的错误调控是复杂疾病的基础，包括癌症、心脏病和神经病理。为了了解miRNAs在正常和疾病条件下的作用，必须解决两个基本问题：(1)miRNA的表达是如何调节的，在疾病状态下是如何改变的；(2)什么是生物学上相关的miRNA靶点，其错误调节导致疾病表型？这些问题是本文提出的工作的基础，目的是阐明控制miRNA生物发生的新机制，并确定miRNA复合体如何识别和调节体内的特定靶点。通常，miRNAs会将ArgAerte(AGO)及其辅助因子招募到特定的mRNAs中，以触发衰退或翻译抑制。在这里，将研究AGO在调节miRNA生物发生中的一个全新角色。在目标1中，AGO用来增强let-7以及可能的其他miRNAs处理的机制将在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的波动具有广泛的相关性。目标2将解决一个令人望而生畏的问题，即不完美的碱基配对如何满足miRNAs对特定靶标的调控。三种互补的方法将被用来破译AGO如何在内生背景下识别和调节特定的目标。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线虫中特定miRNAs引导的AGO的全基因组靶向特性。还将分析AGO与其靶标结合的后果，以全面了解miRNA在活体动物中的功能。最终，这些研究将产生前所未有的数据集，用于破译体内用于多细胞动物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.