Structural and Functional Analysis of Human DICER1

提案摘要/摘要 DICER 1是一种多结构域蛋白，其含有两个RNase III结构域（RNase IIIa和IIIb），所述RNase III结构域催化DICER 1的降解。 前体微小RNA的最终切割以形成成熟微RNA的3 p（3'引物末端）和5 p（5'引物末端）链， microRNA（miRNA）。虽然已经进行了一些初步表征以显示DICER 1如何结合 RNA酶III结构域与pre-miRNA底物相互作用的确切机制 在pre-miRNA切割过程中的作用仍然很不清楚。造成这种知识差距的主要原因之一是， DICER 1的高分辨率（亚3.5 μ m）结构可用于构建详细说明如何 该蛋白质接合并加工前体miRNA底物。 最近的生物化学研究表明，RNase IIIb中关键金属结合残基的点突变 在许多DICER 1综合征患者中常见的结构域，导致有缺陷的miRNA加工。因此，在本发明中， 这些突变为从结构上理解pre-miRNAs如何与RNase III结合提供了独特的策略 结构域而不干扰完全裂解反应。此外，加纳实验室最近的工作 发现了不同种类的天然产物，可用于抑制DICER 1介导的前体miRNA 成熟因此，这些新发现的化合物也可以用作调节DICER 1的化学工具。 活性，并作为结构和功能表征DICER 1相互作用的额外策略 一个pre-miRNA。 这项研究计划的目的是表征与人DICER 1结合的结构-功能关系， 前体miRNA底物在miRNA生物发生中的作用。本提案的具体目标是确定如何 由于RNase III结构域中的突变引起的改变可用于将DICER 1锁定在不同的构象， 以及利用天然产物来调节前体miRNA相互作用。这项工作是基于 DICER 1在miRNA表达过程中必须在RNase III结构域发生关键构象变化的假设 切割，以使pre-miRNA底物适当地朝向与关键的 在催化活性部位发现的金属结合残基。 为了评估我的假设，我将结合联合收割机冷冻电镜结构研究以及前体miRNA切割， 结合试验来研究支配miRNA生物发生的潜在机制。此外，这项工作将 揭示了DICER 1中的关键结合口袋，可以使用小分子和天然药物靶向。 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.