Analysis of the Molecular Machinery of microRNA-processing pathways

MicroRNAs(MiRNAs)是一种22个核苷酸的非编码小RNA，调节翻译、去烯基化 以及它们的目标核糖核酸的衰变。MiRNAs最近在生物学中占据了中心舞台，因为它们的 在动物发育、人类疾病和癌症方面发挥重要作用。 通常，两个RNaseIII家族成员DROSHA和DICER处理miRNA的茎环结构 前驱体顺序排列。我最近的工作揭示了一种新的加工途径，在这种途径中，miRNA成熟 跳过Disher步骤，进入另一条依赖Ago2的途径。这一发现挑战了一个 长期以来，人们认为DICER对miRNA的成熟是必不可少的。然而，Ago2介导的卵裂只是 这一新途径的初始步骤，对其分子机制和机制知之甚少 这是miRNA成熟的最后步骤的基础。我的初步结果表明，在Ago2介导的 裂解，miR-451被尿苷基化，并通过核外溶解修剪加工成成熟形式，这是一种机制 最近有报道称，这是一种广泛使用的提纯成熟miRNA长度的方法。是否修剪 尿酸化机制在规范的和依赖于Ago2的miRNA加工途径中都是共同的 是我将在这个项目中探索的一个问题。 要揭示miRNA修剪(目标1)和尿苷基化机制(目标2)，在指导阶段 大奖I将重点分析miR-451的处理，因为这种处理依赖于Ago2 MiRNA需要普遍的尿苷基化和广泛的修剪。此外，miR-451在 脊椎动物，在红细胞成熟和代谢调节中发挥重要作用 胶质母细胞瘤使得这一项目的结果与人类治疗学相关。 稍后，在该奖项的独立期间，我将利用尿苷基化和 修剪机制分析它们不仅在依赖于Ago2的miRNA生产中的作用，而且在典型的 脊椎动物发育过程中小RNA的生物发生和周转(目标3)。斑马鱼胚胎的使用将是 以全基因组的方式进行这些分析是至关重要的。斑马鱼模式生物的应用 取消了使用单一组织或细胞类型的限制，这些组织或细胞类型的小RNA谱系有限。至 完成所有这些目标我将结合生物化学、质谱学、遗传学和高通量 测序。 这项提案中的实验将确定一种进化保守的机器来处理小的 脊椎动物中的调控RNA。从这个项目中得出的结果将有助于理解 在早期，通过拖尾和剪裁对3‘端的普遍修饰影响体内miRNA靶标的选择 胚胎发生。MiRNAs在早期胚胎发育中发挥关键作用，清除母体mRNAs和 控制miRNA周转的机制将对胚胎发育产生直接影响。此外， 鉴于修剪在miR-451处理中的相关性，基础机械的发现将 潜在地为人类造血系统疾病和癌症的治疗干预奠定了基础。

microRNAs (miRNAs) are 22 nucleotide small non-coding RNAs that regulate translation, deadenylation and decay of their target mRNAs. miRNAs have recently taken central stage in biology due to their fundamental roles in animal development, human disease and cancer. Typically, two RNase III family members, Drosha and Dicer, process the stem-loop structure of miRNA precursors sequentially. My recent work has uncovered a novel processing pathway where miRNA maturation skips the Dicer step and instead enters into an alternative Ago2-dependent pathway. This finding challenges a long-held assumption that Dicer is essential for miRNA maturation. However, Ago2-mediated cleavage is just the initial step of this novel pathway and little is known about the molecular machinery and the mechanisms underlying the final steps of miRNA maturation. My preliminary results suggest that after Ago2-mediated cleavage, miR-451 is uridylated and processed to its mature form by exonucleolytic trimming, a mechanism that has recently been reported as a widespread method to refine mature miRNA length. Whether the trimming and uridylation machinery is common to both the canonical and Ago2-dependent miRNA processing pathways is a question that I will explore in this project. To uncover the miRNA trimming (Aim 1) and uridylation machinery (Aim 2), during the mentored phase of the award I will focus on the analysis of miR-451 processing because processing of this Ago2-dependent miRNA requires pervasive uridylation and extensive trimming. Moreover, miR-451 is conserved across vertebrates and plays an essential role both in erythrocyte maturation and in metabolic regulation of glioblastoma tumors making the results of this project relevant for human therapeutics. Later, in the independent period of the award I will capitalize on the discovery of the uridylation and trimming machinery to analyze their role not only in Ago2-dependent miRNA production but also in canonical small RNA biogenesis and turnover during vertebrate development (Aim 3). The use zebrafish embryos will be crucial to perform these analyses in a genome-wide manner. The use of the zebrafish model organism removes the restrictions of using single tissue or cell types, which have limited repertoire of small RNAs. To accomplish all of these objectives I will combine biochemistry, mass spectrometry, genetics and highthroughput sequencing. The experiments in this proposal will identify an evolutionarily conserved machinery to process small regulatory RNAs in vertebrates. The results derived form this project will be instrumental to understand how pervasive modifications of the 3’-end by tailing and trimming affects miRNA-target selection in vivo during early embryogenesis. miRNAs play a key role during early embryogenesis clearing maternal mRNAs and mechanisms controlling miRNA turnover will have a direct impact in embryonic development. Furthermore, given the relevance of trimming in miR-451 processing, the discovery of the underlying machinery will potentially set the framework for therapeutic intervention in human hematopoietic disorders and cancer.