Analysis of the Molecular Machinery of microRNA-processing pathways

microRNAs（miRNAs）是一类由22个核苷酸组成的小分子非编码RNA，它们调节翻译、去腺苷化、 和它们的靶mRNA的衰变。最近，由于它们的功能，miRNA在生物学中占据了中心地位。 在动物发育、人类疾病和癌症中的重要作用。 通常，两个RNase III家族成员，Drosha和Dicer，处理miRNA的茎环结构 前体顺序我最近的工作揭示了一种新的加工途径， 跳过Dicer步骤，而是进入替代的Ago 2依赖性途径。这一发现挑战了 Dicer对miRNA成熟至关重要。然而，Ago 2介导的切割只是 这一新途径的初始步骤，而对分子机制和机制知之甚少 这是miRNA成熟的最后一步。我的初步结果表明，在Ago 2介导的 切割后，miR-451被尿苷酸化，并通过核酸外切酶修整加工成其成熟形式，这是一种机制， 这是最近报道的一种广泛使用的细化成熟miRNA长度的方法。无论是修剪 尿苷酸化机制在经典和Ago 2依赖的miRNA加工途径中是共同的 这是我在这个项目中要探讨的问题。 为了揭示miRNA修剪（Aim 1）和尿苷化机制（Aim 2），在指导阶段， 该奖项我将重点分析miR-451的加工，因为这种Ago 2依赖的加工 miRNA需要普遍的尿苷化和广泛的修剪。此外，miR-451在整个细胞中是保守的。 在脊椎动物中，在红细胞成熟和代谢调节中起重要作用。 胶质母细胞瘤肿瘤使得该项目的结果与人类治疗相关。 后来，在该奖项的独立期间，我将利用尿苷化的发现， 修剪机器，以分析它们不仅在Ago 2依赖性miRNA产生中的作用，而且在典型的 脊椎动物发育过程中的小RNA生物合成和周转（目的3）。利用斑马鱼胚胎将 关键是以全基因组的方式进行这些分析。斑马鱼模式生物的应用 消除了使用单一组织或细胞类型的限制，这些组织或细胞类型具有有限的小RNA库。到 为了实现所有这些目标，我将联合收割机结合生物化学，质谱，遗传学和高通量 测序 这项提议中的实验将确定一种进化上保守的机制， 在脊椎动物中的调节RNA。从这个项目得出的结果将有助于了解如何 通过加尾和修剪对3 '末端的普遍修饰影响早期miRNA的体内靶选择， 胚胎发生miRNAs在早期胚胎发生过程中起关键作用，清除母体mRNAs， 控制miRNA周转的机制将对胚胎发育产生直接影响。此外，委员会认为， 考虑到修剪在miR-451加工中的相关性，潜在机制的发现将 可能为人类造血系统疾病和癌症的治疗干预建立框架。

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.