Analysis of the Molecular Machinery of microRNA-processing pathways

microRNA 加工途径的分子机械分析

• 批准号: 8442460
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8442460
• 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; 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（申请人提供）：关于项目：microRNAs（miRNAs）是22个核苷酸的小非编码RNA，调节其靶mRNA的翻译，去腺苷化和衰变。由于其在动物发育、人类疾病和癌症中的基本作用，miRNAs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。我最近的工作揭示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Ago 2依赖性途径。这一发现挑战了长期以来的假设，即Dicer对于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，并且对Ago 2介导的切割机制知之甚少。 分子机制和miRNA成熟的最后步骤的机制。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剪加工成成熟形式，这是一种最近被报道为细化成熟miRNA长度的广泛方法的机制。是否修剪和尿苷化机制是共同的经典和Ago 2依赖的miRNA加工途径是一个问题，我将在这个项目中探索。 为了揭示miRNA修剪（Aim 1）和尿苷化机制（Aim 2），在该奖项的指导阶段，我将重点分析miR-451加工，因为这种Ago 2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肿瘤的代谢调节中起重要作用，使得该项目的结果与人类治疗相关。 稍后，在该奖项的独立期间，我将利用尿苷酸化和修剪机制的发现来分析它们不仅在Ago 2依赖的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（Aim 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。斑马鱼模式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具有有限的小RNA库。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。 在这个建议中的实验将确定一个进化上保守的机器来处理脊椎动物中的小调控RNA。来自该项目的结果将有助于理解在早期胚胎发生期间，通过加尾和修剪对3 '端的普遍修饰如何影响体内miRNA靶向选择。miRNA在早期胚胎发生过程中发挥关键作用，清除母体mRNA，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。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助理培训，在那里他将享受最先进的设施，并与他的导师和该领域的其他科学领导人进行互动。在这种环境下，Cifuentes博士将利用职业发展奖来实现他的短期目标，即在i）计算生物学和ii）质谱学领域获得特定培训。这将使他能够获得高质量的出版物，并在microRNA领域独立于他以前的导师。这些成就将建立框架，以实现理解替代加工途径如何调节miRNA功能以及这些途径在发育和人类疾病中的影响的长期目标。此外，Cifuentes博士计划通过参加结构化课程和研讨会来扩大他以前的教学和指导经验。这将使他能够学习创新和有效的方法来教授生物学和进步，成为一个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职的任命。Cifuentes博士将在完成职业发展奖后有竞争力地获得此类小组领导人的任命，这是为提高他的科学和指导技能而量身定制的。