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个核苷酸的非编码小RNA，调节其目标mRNAs的翻译、去烯化和衰变。由于在动物发育、人类疾病和癌症中的基础作用，miRNAs最近在生物学中占据了中心地位。通常，两个RNaseIII家族成员，DROSHA和DICER，顺序地处理miRNA前体的茎环结构。我最近的工作发现了一条新的处理途径，其中miRNA成熟跳过了Disher步骤，而进入了另一条依赖Ago2的途径。这一发现挑战了长期以来的假设，即迪格尔对于miRNA的成熟是必不可少的。然而，Ago2介导的切割只是这一新途径的第一步，人们对Ago2介导的切割还知之甚少 分子机制和miRNA成熟的最后步骤的潜在机制。我的初步结果表明，在Ago2介导的切割后，miR-451被尿苷化，并通过核外切割加工成成熟的形式，这一机制最近被报道为一种广泛应用的提纯成熟miRNA长度的方法。剪裁和尿苷酸化机制是否对规范的和依赖于Ago2的miRNA加工途径是共同的，这是我将在这个项目中探索的问题。为了揭示miRNA修剪(目标1)和尿苷化机制(目标2)，在奖项的指导阶段，我将重点分析miR-451的加工，因为这种依赖于Ago2的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，在红细胞成熟和胶质母细胞瘤肿瘤的代谢调节中发挥着重要作用，使该项目的结果与人类治疗相关。稍后，在该奖项的独立期，我将利用尿基化和修剪机制的发现，不仅分析它们在依赖Ago2的miRNA生产中的作用，而且分析它们在脊椎动物发育过程中典型的小RNA生物发生和周转中的作用(目标3)。斑马鱼胚胎的使用将是以全基因组方式进行这些分析的关键。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理解在早期胚胎发育过程中，通过拖尾和剪裁对3‘端的普遍修改如何影响体内miRNA靶标的选择。MiRNAs在早期胚胎发育过程中起着关键作用，清除母体mRNAs和控制miRNA周转的机制将直接影响胚胎发育。此外，考虑到修剪在miR-451加工过程中的相关性，潜在机制的发现将可能为人类造血疾病和癌症的治疗干预奠定框架。候选人简介：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，并与他的导师和该领域的其他科学领袖进行互动。在这种环境下，Cifuentes博士将利用职业发展奖来实现他在i)计算生物学和ii)质谱学领域获得特定培训的短期目标。这将使他能够获得高质量的出版物和在microRNA领域的强大存在，而不依赖于他以前的导师。这些成就将建立框架，以实现了解替代加工途径如何调节miRNA功能以及这些途径在发育和人类疾病中的影响这一长期目标。此外，Cifuentes博士计划通过参加有组织的课程和研讨会来扩大他以前的教学和指导经验。这将使他学习创新和有效的方法来教授生物学，并进步成为一名更全面的科学家和导师。西富恩特斯博士完全致力于获得学术界终身教职的任命。完全可以预期的是，Cifuentes博士将在他的职业发展奖结束时竞争这些小组领导人的任命，该奖项是为提高他的科学和指导技能而量身定做的。