Structural and Functional Analysis of Human DICER1

建议书摘要/摘要 DICER1是一种多结构域蛋白质，它包含两个RNaseIII结构域(RNaseIIIa和RNaseII1b)，它们催化 对前microRNA的最终切割，形成成熟的3p(3‘末端)和5p(5’末端)链 MicroRNA(MiRNA)。虽然已经做了一些初步表征来显示DICER1是如何结合的 对于前miRNA底物，RNaseIII结构域与前miRNA相互作用的确切机制 在miRNA前切割过程中，很大程度上仍不清楚。这种知识差距背后的主要原因之一是 没有DICER1的高分辨率(低于3.5？)结构可用于构建详细说明如何 该蛋白质与miRNA前底物结合并加工。 最近的生化研究表明，RNaseIIIb中关键金属结合残基的点突变 结构域在许多DICER1综合征患者中很常见，导致miRNA处理缺陷。因此， 这些突变为从结构上理解前miRNAs如何与RNaseIII结合提供了一种独特的策略 结构域不受完全切割反应的干扰。此外，加纳实验室最近的工作已经导致了 发现可用于抑制DICER1介导的Pre-miRNA的不同类别的天然产物 成熟。因此，这些新发现的化合物也可以作为化学工具来调节DICER1 活动，并作为从结构和功能上表征DICER1相互作用的额外策略 带有前miRNA。 本研究的目的是刻画与DICER1结合的人DICER1的结构功能关系 MiRNA生物发生过程中的前miRNA底物。这项提案的具体目标是确定如何 RNaseIII结构域的突变可用于将DICER1锁定在不同的构象中 并利用天然产物来调节miRNA前相互作用。这项工作是基于 假设DICER1在miRNA过程中必须经历RNaseIII结构域的关键构象变化 切割，以便适当地将前-miRNA底物定向到与临界 在催化活性中心发现金属结合残基。 为了评估我的假设，我将结合冷冻-EM结构研究以及前miRNA切割和 结合分析是研究miRNA生物发生的潜在机制。此外，这项工作将 揭示了DICER1中可以使用小分子和天然分子作为靶点的键绑定口袋 产品作为治疗miRNA相关疾病的替代方法。

PROPOSAL SUMMARY/ABSTRACT DICER1 is a multidomain protein that contains two RNase III domains (RNase IIIa and IIIb) that catalyze the final cleavage of a pre-microRNA to form the 3p (3' prime end) and 5p (5' prime end) strands of a mature microRNA (miRNA), respectively. While some initial characterization has been done to show how DICER1 binds to a pre-miRNA substrate, the exact mechanism by which the RNase III domains interact with the pre-miRNA during pre-miRNA cleavage is still largely unclear. One of the major causes behind this gap in knowledge is that no high-resolution (sub-3.5Å) structures of DICER1 are available for building a molecular model detailing how the protein engages and processes pre-miRNA substrates. Recent biochemical studies have shown that point mutations at key metal-binding residues in the RNase IIIb domain, which are common in many DICER1 Syndrome patients, cause defective miRNA processing. thus, these mutations offer a unique strategy to structurally understand how pre-miRNAs binds to the RNase III domains without interference of the full cleavage reaction. Additionally, recent work from the Garner lab has led to the discovery of distinct classes of natural products that can be used to inhibit DICER1-mediated pre-miRNA maturation. Thus, these newly discovered compounds can also be used as chemical tools to modulate DICER1 activity, and as an additional strategy by which to structurally and functionally characterize DICER1 interactions with a pre-miRNA. This research proposal aims to characterize the structure-function relationship of human DICER1 bound to pre-miRNA substrates during miRNA biogenesis. The specific objective of this proposal is to determine how alterations due to mutations in the RNase III domains can be used to lock DICER1 in different conformational states, as well as to utilize natural products to modulate pre-miRNA interactions. This work is based on the hypothesis that DICER1 must undergo key conformational changes in the RNase III domains during miRNA cleavage in order to properly orient the pre-miRNA substrate towards a favorable interaction with the critical metal binding residues found in the catalytic active site. To evaluate my hypothesis, I will combine cryo-EM structural studies as well as pre-miRNA cleavage and binding assays to study the underlying mechanism that dictates miRNA biogenesis. Furthermore, this work will shed new light on key binding pockets within DICER1 that can be targeted using small molecules and natural products as an alternative approach to therapeutically treating miRNA linked diseases.