Molecular Characterization of the microRNA Processing Pathways

描述（由申请人提供）：微小RNA（miRNA）编码~ 22 nt小RNA，其调节其靶mRNA的去腺苷化、翻译和衰变。由于有可能调节超过30%的人类基因，miRNA在从人类发育到人类疾病（包括神经精神疾病和癌症）的生物学各个方面发挥着重要作用。在动物中，miRNA来源于由两种连续的RNAse III酶Drosha和Dicer加工的特征发夹。我们最近的工作已经确定了一种新的microRNA加工途径，它独立于Dicer功能，依赖于Argonaute 2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，支配Dicer与Argonaute加工的规则以及该途径的下游组分在很大程度上仍然未知。 该提案结合了生物化学、质谱遗传学和高通量测序，旨在了解决定进入Argonaute与Dicer加工途径的结构和序列因素（Aim 1），通过Argonaute切割的中间体的修剪和尿苷化，鉴定Argonaute 2下游产生成熟miRNA所需的机制（Aim 2）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加工的异常与发育缺陷和人类癌症有关。特别是，miR-451是一种仅由Argonaute 2加工的microRNA，与人类神经胶质瘤形成和血液疾病相关。因此，Argonaute 2加工下游所需机制的鉴定将有助于我们了解microRNA加工功能障碍如何导致人类出生缺陷并导致疾病。 总之，所提出的实验挑战了本领域的经典观点，即所有microRNA都被Dicer加工，其长期目标是i）提供脊椎动物发育期间小RNA的加工、序列和基因组起源的深入表征，提供理解其体内功能的切入点，和ii）揭示脊椎动物中加工小调节RNA所需的进化上保守的机制，解决小RNA加工、基因调节、RNA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.