Mechanisms of Microprocessor Function and Regulation

项目概要/摘要（30行） microRNAs（miRNAs）是一类短的、非编码的、调控蛋白质表达的小分子RNA 表情异常的miRNA水平与许多疾病有关，包括发育缺陷和 各种癌症。为了产生功能性miRNA，初级转录物（pri-miRNAs）通常需要首先被转录。 被核糖核酸酶III切割了卓沙这一关键步骤的miRNA生物合成需要控制，在这两个 准确性和效率，以维持适当的基因调控。加工酶Drosha需要其 配偶体蛋白DGCR8用于蛋白稳定性和底物特异性。Drosha分子结合同源二聚体 DGCR8进行pri-miRNA加工，但也可能形成更高级的复合物，因为DGCR8的聚集 基因组中的pri-miRNAs增强了加工。我们最近突破性的低温电子显微镜（cryo- EM）结构提供了作用中的微处理器-初级-miRNA复合物的原子模型。阐述了 蛋白质组织在RNA茎环周围，也揭示了每个远端（基底或顶端）是如何 它们被独立地识别，但也通过连接检测模块的分子标尺彼此连接。 拟议的研究建立在我们之前的成功基础上，因为结构框架将使我们能够获得 对大分子识别是如何完成的新的基本见解。我们的总体目标是 理解微处理器在分子水平上识别pri-miRNAs。我们假设RNA 结构特征和上下文依赖的序列偏好决定了每个个体的加工命运 pri-miRNA我们将剖析识别是如何完成在每一个基层和顶端交界处的初级， miRNAs。我们还将研究来自不同pri-miRNAs的不同RNA序列和结构如何影响 正确识别每个连接点，以揭示多部分机器的可塑性。我们之前的工作 这也让我们准备好解决紧迫的问题，即pri-miRNAs的聚集如何通过以下方式增强加工： 微处理器，这是至关重要的，我们的整体理解的miRNA生物发生，并具有深刻的 在各种各样的领域获得的以前和未来的结果的解释的影响， 对miRNA基因的操作。更好地把握核心识别机制将有助于我们解释独特的 例如成簇的pri-miRNAs。建议的研究将提供全面的 理解miRNAs的加工和调节，miRNAs是基因表达的重要调节因子。我们的工作 破译结构如何影响RNA识别是一个根本性的重要问题， 对许多涉及结构化RNA而非miRNA的过程具有深刻的见解。

Project Summary/Abstract (30 lines) MicroRNAs (miRNAs) constitute a large family of short, non-coding, regulatory RNAs that modulate protein expression. Abnormal miRNA levels are associated with many diseases, including developmental defects and various cancers. To generate functional miRNAs, primary transcripts (pri-miRNAs) generally need to be first cleaved by an RNaseIII, Drosha. This critical step of miRNA biogenesis needs to be controlled, in both accuracy and efficiency, to maintain proper gene regulation. The processing enzyme Drosha requires its partner protein, DGCR8, for protein stability and substrate specificity. A Drosha molecule binds homo-dimeric DGCR8 to carry out pri-miRNA processing, but higher-order complexes may also form because clustering of pri-miRNAs in the genome enhances processing. Our recent groundbreaking cryo-electron microscopy (cryo- EM) structures provide atomic models of the Microprocessor-pri-miRNA complex in action. Elucidating how the proteins are organized around the RNA stem-loop also revealed how each distal end (basal or apical) is independently recognized but also linked to each other via a molecular ruler connecting the detection modules. The proposed research builds on our previous successes, as the structural framework will enable us to gain novel fundamental insights into how the macromolecular recognition is accomplished. Our overall goal is to understand the recognition of pri-miRNAs by Microprocessor at the molecular level. We hypothesize that RNA structural features and context-dependent sequence preferences dictate the processing fate of each individual pri-miRNA. We will dissect how the recognition is accomplished at each of the basal and apical junctions of pri- miRNAs. We will also investigate how diverse RNA sequences and structures from different pri-miRNAs affect proper recognition at each junction, to reveal the plasticity of the multipart machinery. Our previous work has also left us poised to address urgent questions on how clustering of pri-miRNAs enhances processing by Microprocessor, which is crucial for our overall understanding of miRNA biogenesis and has profound implications for the interpretation of previous and future results in a wide variety of fields obtained by manipulation of miRNA genes. A better grasp of the core recognition mechanisms will help us explain unique targets such as clustered pri-miRNAs. Together, the proposed studies will provide a comprehensive understanding of processing and regulation of miRNAs, important regulators of gene expression. Our work on deciphering how structure affects RNA recognition is a fundamentally important question and likely be insightful for many processes involving structured RNAs beyond miRNAs.