Molecular Characterization of the microRNA Processing Pathways

描述(申请人提供)：microRNAs(MiRNAs)编码~22nt的小RNA，调节其目标mRNAs的去烯基化、翻译和衰变。MiRNAs有可能调控超过30%的人类基因，在生物学的各个方面发挥着重要作用，从人类发育到包括神经精神障碍和癌症在内的人类疾病。在动物中，miRNAs来自两种顺序的RNaseIII酶DROSHA和DICER加工的特征发夹。我们最近的工作发现了一条新的不依赖于Disher功能的microRNA加工途径，该途径依赖于ArgAerte2的催化活性作为初始加工步骤，并且是红细胞发育所必需的。然而，管理Disher和ArgAerte加工以及这一途径的下游组件的规则在很大程度上仍不清楚。该建议结合了生物化学、质谱学遗传学和高通量测序，目的是了解决定ArgAerte与Diller加工途径(Aim 1)进入的结构和序列因素，确定ArgAerte2下游通过修剪和尿基化ArgAerte切割的中间体(Aim 2)产生成熟miRNA所需的机械，并以斑马鱼为模型系统确定脊椎动物发育过程中所有microRNA的加工要求(Aim 3)。MicroRNA加工过程中的异常与发育缺陷和人类癌症有关。特别是，miR-451是一种由ArgAerte2独家处理的微RNA，与人类胶质瘤的形成和血液疾病有关。因此，识别ArgAerte2加工下游所需的机械将有助于我们了解microRNA加工功能障碍可能如何导致人类出生缺陷并导致疾病。总之，拟议的实验挑战了该领域的经典观点，即所有的microRNAs都由DICER处理，其长期目标是：i)提供脊椎动物发育过程中小RNA的处理、序列和基因组起源的深入表征，为了解它们在体内的功能提供一个切入点；以及ii)揭示在脊椎动物中处理小调控RNA所需的进化保守的机制，解决小RNA处理、基因调控、RNA新陈代谢中的基本问题。

DESCRIPTION (provided by applicant): microRNAs (miRNAs) encode ~22nt small RNAs that regulate deadenylation, translation, and decay of their target mRNAs. With the potential to regulate more that 30% of the human genes, miRNAs play fundamental roles in every aspect of biology from human development, to human disease including neuropsychiatric disorders and cancer. In animals, miRNAs are derived from characteristic hairpins processed by two sequential RNAse III enzymes, Drosha and Dicer. Our recent work has identified a novel microRNA processing pathway independent of Dicer function that depends on the catalytic activity of Argonaute2 as the initial processing step and is required for red blood cell development. Yet, the rules that govern Dicer vs. Argonaute processing and the downstream components of this pathway remain largely unknown. This proposal combines, biochemistry, mass spectrometry genetics and high-throughput sequencing with the aim to understand the structural and sequence factors that determine entry in the Argonaute vs. the Dicer processing pathway (Aim 1), identify the machinery downstream of Argonaute2 required to generate the mature miRNA through trimming and uridylation of the argonaute cleaved intermediate (Aim 2) and identify the processing requirements for all microRNAs during vertebrate development (Aim 3) using zebrafish as a model system. Abnormalities in microRNA processing have been associated with developmental defects and human cancer. In particular, miR-451 a microRNA exclusively processed by Argonaute2, is associated with glioma formation and blood disorders in humans. Thus, the identification of the machinery required downstream of Argonaute2 processing will help us understand how the dysfunction of microRNA processing might cause human birth defects and contribute to disease. In summary, the proposed experiments challenge a classical view in the field that all microRNAs are processed by Dicer have the long term goal of i) providing in-depth characterization of the processing, sequence and genomic origin of small RNAs during vertebrate development, providing an entry point to understand their function in vivo, and ii) uncovering an evolutionarily conserved machinery required to process small regulatory RNAs in vertebrates addressing fundamental questions in small RNA processing, gene regulation, RNA metabolism.