Analysis of the Molecular Machinery of microRNA-processing pathways

microRNA 加工途径的分子机械分析

• 批准号: 8698432
• 负责人: Daniel Cifuentes
• 金额: $ 10.87万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-08 至 2015-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8698432
• 关键词: 5' -exoribonuclease; Academia; Address; Affect; Alleles; Animal Model; Animals; Appointment; Area; Award; Biochemical; Biochemistry; Biogenesis; Biology; Bypass; Cells; Commit; Computational Biology; Data; Defect; Development; Disease; Educational process of instructing; Educational workshop; Embryo; Embryonic Development; Environment; Enzymes; Erythrocytes; Exonuclease; Faculty; Family member; Fishes; Functional RNA; Gene Expression; Genes; Genetic; Glioblastoma; Goals; Hematological Disease; Hematopoietic; High-Throughput Nucleotide Sequencing; Histocompatibility Testing; Human; Human Development; Introns; K-Series Research Career Programs; Knowledge; Lead; Learning; Length; Link; Malignant Neoplasms; Mammalian Cell; Mass Spectrum Analysis; Maternal Messenger RNA; Mediating; Mentors; Messenger RNA; Metabolic; Methods; MicroRNAs; Modification; Molecular; Molecular Analysis; Nucleotides; Pathway interactions; Phase; Phenotype; Phosphodiesterase I; Play; Population; Process; Production; Proteins; Publications; RNA Processing; Regulation; Regulator Genes; Reporting; Research; Ribonuclease III; Role; Scientist; Signal Transduction; Site; Small Nucleolar RNA; Small RNA; Staging; Structure; Tail; Techniques; Testing; Therapeutic; Therapeutic Intervention; Training; Transfer RNA; Translations; UDPglucose-Hexose-1-Phosphate Uridylyltransferase; Universities; Vertebrates; Work; Zebrafish; cell type; crosslink; developmental disease; experience; genome-wide; genome-wide analysis; human DICER1 protein; human disease; in vivo; innovation; insight; loss of function; mutant; novel; public health relevance; research study; skills; stem; tumor

DESCRIPTION (provided by applicant): About the project: 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 of 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 high- throughput sequencing. The experiments in this proposal will identify an evolutionarily conserved machinery to process small regulatory RNAs in vertebrates. The results derived from 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. About the candidate: Dr. Cifuentes will continue his training as Postdoctoral associate in the Genetics Department at Yale University, where he will enjoy both state-of-the-art facilities and the interaction with his mentors and other scientific leaders of th field. In this environment, Dr. Cifuentes will use the Career Development Award to fulfill his short-term goal of acquiring specific training in the areas of i) computational biology and ii) mass- spectrometry. This will allow him to obtain high quality publications and a strong presence in the microRNA field independent of his previous mentors. These accomplishments will establish the framework to achieve the long-term goal of understanding how alternative processing pathways modulate miRNA function and the impact of these pathways in development and human disease. Furthermore, Dr. Cifuentes plans to expand his previous teaching and mentoring experience by participating in structured courses and workshops. This will allow him to learn innovative and effective ways to teach biology and progress to become a more rounded scientist and mentor. Dr. Cifuentes is fully committed to obtain an appointment as a tenure track junior faculty in academia. It is fully expected that Dr. Cifuentes will be competitive for such group leader appointments at the completion of his Career Development Award, tailored to enhance his scientific and mentoring skills.

描述（由申请人提供）：关于该项目：microRNA (miRNA) 是 22 个核苷酸的小非编码 RNA，可调节其目标 mRNA 的翻译、去腺苷酸化和衰变。由于 miRNA 在动物发育、人类疾病和癌症中的重要作用，miRNA 最近在生物学中占据了中心地位。 通常，两个 RNase III 家族成员 Drosha 和 Dicer 依次处理 miRNA 前体的茎环结构。我最近的工作发现了一种新的加工途径，其中 miRNA 成熟跳过 Dicer 步骤，而是进入另一种依赖 Ago2 的途径。这一发现挑战了长期以来的假设，即 Dicer 对于 miRNA 的成熟至关重要。然而，Ago2 介导的裂解只是这一新途径的第一步，人们对它知之甚少。 miRNA 成熟最终步骤的分子机制和机制。我的初步结果表明，在 Ago2 介导的切割后，miR-451 被尿苷化并通过核酸外切修剪加工成成熟形式，这种机制最近被报道为细化成熟 miRNA 长度的广泛方法。修剪和尿苷化机制是否对于规范和 Ago2 依赖的 miRNA 加工途径是通用的，这是我将在本项目中探讨的一个问题。 为了揭示 miRNA 修剪（目标 1）和尿苷化机制（目标 2），在该奖项的指导阶段，我将重点分析 miR-451 加工，因为这种 Ago2 依赖性 miRNA 的加工需要普遍的尿苷化和广泛的修剪。此外，miR-451在脊椎动物中是保守的，并且在红细胞成熟和胶质母细胞瘤的代谢调节中发挥重要作用，使得该项目的结果与人类治疗相关。 随后，在该奖项的独立阶段，我将利用尿苷化和修剪机制的发现来分析它们不仅在 Ago2 依赖性 miRNA 产生中的作用，而且在脊椎动物发育过程中的典型小 RNA 生物发生和周转中的作用（目标 3）。斑马鱼胚胎的使用对于以全基因组方式进行这些分析至关重要。斑马鱼模型生物的使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的小 RNA 库有限。为了实现所有这些目标，我将结合生物化学、质谱、遗传学和高通量测序。 该提案中的实验将确定一种进化上保守的机制来处理脊椎动物中的小调控 RNA。该项目的结果将有助于了解通过加尾和修剪对 3' 端进行普遍修饰如何影响早期胚胎发生过程中体内 miRNA 靶标的选择。 miRNA 在早期胚胎发生过程中发挥着关键作用，清除母体 mRNA 和控制 miRNA 周转的机制将对胚胎发育产生直接影响。此外，考虑到 miR-451 加工中修剪的相关性，潜在机制的发现将有可能为人类造血疾病和癌症的治疗干预奠定框架。关于候选人：Cifuentes 博士将继续在耶鲁大学遗传学系接受博士后培训，在那里他将享受最先进的设施以及与导师和该领域其他科学领袖的互动。在这种环境下，Cifuentes 博士将利用职业发展奖来实现他在 i) 计算生物学和 ii) 质谱领域获得特定培训的短期目标。这将使他能够独立于之前的导师而获得高质量的出版物并在 microRNA 领域拥有强大的影响力。这些成就将建立一个框架，以实现了解替代加工途径如何调节 miRNA 功能以及这些途径对发育和人类疾病的影响的长期目标。此外，Cifuentes 博士计划通过参加结构化课程和研讨会来扩展他之前的教学和指导经验。这将使他能够学习创新且有效的生物学教学方法，并成为一名更全面的科学家和导师。 Cifuentes 博士完全致力于获得学术界终身教授职位。完全预计 Cifuentes 博士在获得职业发展奖后将能够竞争此类小组领导者的任命，该奖是为提高他的科学和指导技能而量身定制的。