Molecular Characterization of the microRNA Processing Pathways

描述（由申请人提供）：microRNAs （miRNAs）编码约22nt小rna，调节其靶mrna的死基化，翻译和衰变。mirna有可能调节超过30%的人类基因，从人类发育到包括神经精神疾病和癌症在内的人类疾病，mirna在生物学的各个方面都发挥着重要作用。在动物中，mirna来源于两种连续的RNAse III酶Drosha和Dicer处理的特征发夹。我们最近的工作已经确定了一种独立于Dicer功能的新型microRNA加工途径，该途径依赖于Argonaute2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，控制Dicer和Argonaute加工的规则以及这一途径的下游成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Argonaute与Dicer加工途径进入的结构和序列因素（aim 1）。鉴定Argonaute2下游通过argonaute裂解中间体的修剪和尿苷化产生成熟miRNA所需的机制（Aim 2），并以斑马鱼为模型系统鉴定脊椎动物发育过程中所有microrna的加工需求（Aim 3）。microRNA加工异常与发育缺陷和人类癌症有关。特别是，由Argonaute2专门加工的microRNA miR-451与人类胶质瘤的形成和血液疾病有关。因此，鉴定Argonaute2加工下游所需的机制将有助于我们了解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该领域的经典观点，即所有微小rna都是由Dicer加工的，其长期目标是：1)深入表征脊椎动物发育过程中小rna的加工、序列和基因组起源，为了解它们在体内的功能提供切入点；ii)揭示脊椎动物加工小调控RNA所需的进化保守机制，解决小RNA加工、基因调控、RNA代谢等基本问题。

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.