Mechanisms of Microprocessor Function and Regulation

项目概要/摘要（30行） microRNA（miRNAs）是一类短的、非编码的调控RNA。产生功能性 miRNA，初级转录物（pri-miRNA）需要首先被RNA酶III酶Drosha切割。这 miRNA成熟的关键步骤需要严格控制，而失调与许多 疾病加工效率也可以通过各种手段来调整，包括转录后 RNA的修饰，如通过Mettl 3/Mettl 14复合物的腺苷甲基化。此外，德罗沙是 依赖于它的伴侣蛋白DGCR 8。DGCR 8在mRNA和蛋白质水平上受到调节，并且还在mRNA水平上受到调节。 由血红素结合调节。然而，pri-miRNA加工如何进行的机制细节和 调控仍然在很大程度上是未知的，留下了许多问题没有答案：1）是什么使pri-miRNA成为一个好的 Drosha处理的基板？2)RNA修饰的变化是如何调节加工速率的 还是构象3)为什么Drosha需要一个辅因子蛋白（DGCR 8），不像其他RNAseIII酶？ DGCR 8和Drosha如何协同识别、结合和精确切割底物RNA？ 我们的长期目标是在原子水平上了解miRNA的加工和调控。的 Drosha/DGCR 8复合体（也称为微处理器）必须有效地处理数千个不同的 转录本，具有单核苷酸精度。我们广泛的假设是，RNA的特征-序列和 结构决定了每个pri-miRNA的加工命运。我们提出的研究重点是阐明 微处理器如何识别pri-miRNAs并与之相互作用的模型，以及识别pri-miRNAs的特征， 允许特定途径调节加工的miRNAs。我们的具体目标是：1）确定如何 pri-miRNAs的N6-甲基腺苷（m6 A）修饰调节加工效率，从而鉴定了 对微处理器活性重要的pri-miRNA特征; 2）确定DGCR 8在其血红素结合中如何与微处理器活性相关; 3）确定DGCR 8在其血红素结合中如何与微处理器活性相关。 状态帮助Drosha，从而剖析DGCR 8在pri-miRNA加工中的分子作用;和3）揭示 微处理器/pri-miRNA复合物的三维结构来解释生化观察结果 前两个目标。 作为一类致力于基因调控的分子，miRNA涉及生物学的每个主要领域， 几乎所有的miRNAs的成熟都需要微处理器。我们提议的研究将共同 阐明Drosha和DGCR 8如何合作识别pri-miRNAs的某些特征。除了 为微处理器的工作方式提供了一个基本的物理框架，我们所研究的分子机制， 这一发现将为研究调节单个miRNAs的各种方法奠定基础， 开发新的治疗药物来靶向基因调控。 -1-

Project Summary/Abstract (30 lines) MicroRNAs (miRNAs) constitute a large family of short, non-coding, regulatory RNAs. To generate functional miRNAs, primary transcripts (pri-miRNAs) need to be first cleaved by an RNAse III enzyme, Drosha. This critical step of miRNA maturation needs to be strictly controlled, and dysregulation is associated with many diseases. Processing efficiency can also tuned through various means, including post-transcriptional modification of RNA, such as adenosine methylation by Mettl3/Mettl14 complexes. In addition, Drosha is dependent on its partner protein, DGCR8. DGCR8 is regulated at the mRNA and protein levels, and is also regulated by heme binding. However, mechanistic details of how pri-miRNA processing is carried out and regulated remain largely unknown, leaving many questions unanswered: 1) what makes a pri-miRNA a good substrate for processing by Drosha? 2) how are processing rates modulated by changes in RNA modification or conformation? 3) why does Drosha require a cofactor protein (DGCR8), unlike other RNAseIII enzymes? and 4) how do DGCR8 and Drosha cooperate to recognize, bind and precisely cleave substrate RNAs? Our long-term goal is to understand miRNA processing and regulation at the atomic level. The Drosha/DGCR8 complex (also called Microprocessor) must efficiently process thousands of different transcripts, with single-nucleotide precision. Our broad hypothesis is that RNA features—sequence and structure—dictate the processing fate of each individual pri-miRNA. Our proposed study focuses on elucidating a model for how Microprocessor recognizes and interacts with pri-miRNAs, and identifying the features of pri- miRNAs that allow specific pathways to regulate processing. Our specific objectives are to: 1) Determine how N6-methyladenosine (m6A) modification of pri-miRNAs modulates processing efficiency, thereby identifying the pri-miRNA features that are important for Microprocessor activity; 2) determine how DGCR8 in its heme-bound state helps Drosha, thereby dissecting the molecular role of DGCR8 in pri-miRNA processing; and 3) Reveal three-dimensional structures of Microprocessor/pri-miRNA complexes to explain the biochemical observations in the first two Aims. As a class of molecules devoted to gene regulation, miRNAs are involved in every major area of biology, and Microprocessor is required for proper maturation of almost all miRNAs. Our proposed studies will together elucidate how Drosha and DGCR8 cooperate to recognize certain features of pri-miRNAs. In addition to providing a basic physical framework for how Microprocessor works, the molecular mechanisms that we discover will lay the groundwork for investigating various ways to regulate individual miRNAs, opening doors to developing new therapeutic agents to target gene regulation. -1-