Analysis of non-canonical functions of microRNAs

microRNA的非典型功能分析

• 批准号: 10563155
• 负责人: Daniel Cifuentes
• 金额: $ 34.41万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10563155
• 关键词: Acute; Address; Affect; Anemia; Binding; Biochemical; Biogenesis; Bioinformatics; Biological Assay; Biological Models; Blood; CRISPR/Cas technology; Catalytic Domain; Cell Reprogramming; Cells; Data; Development; Disease; Dose; Embryo; Embryonic Development; Ensure; Erythrocytes; Erythroid; Erythropoiesis; Family; Fishes; Flow Cytometry; Future; Gene Expression Profiling; Genes; Genetic; Genetic Models; Genomic approach; Goals; Hematopoietic; Homeostasis; Human; Mediating; Messenger RNA; MicroRNAs; Molecular; Pathway interactions; Process; Production; Repression; Ribonuclease III; Role; Route; Series; Small RNA; Specific qualifier value; Structure; System; Testing; Therapeutic Intervention; Time; Transcript; Untranslated RNA; Work; Zebrafish; aptamer; embryo tissue; experience; experimental study; gene repression; genetic approach; improved; induced pluripotent stem cell; inhibitor; insight; mutant; novel; overexpression; posttranscriptional; progenitor; programs; stem; stem cell differentiation; transcriptome sequencing

PROJECT SUMMARY: “Analysis of non-canonical functions of microRNAs” Commitment to cell-fate decisions is fundamental for proper embryonic development and tissue homeostasis. microRNAs, a family of small non-coding RNAs, are among the factors that actively participate in many differentiation processes. Indeed, the last step of differentiation during erythropoiesis is in part governed by miR- 451 and its demise leads to severe anemia. Surprisingly, miR-451 is the only known microRNA whose processing is Dicer-independent but Ago2-dependent. Paradoxically, Dicer is still expressed in erythrocytes, raising the question of what possible advantage represents for erythropoiesis to process miR-451 through a non-canonical pathway. The goal of this project is to uncover by which mechanisms the non-canonical processing of miR-451 becomes indispensable for erythrocyte differentiation. We hypothesize that this alternative processing pathway favors miR-451 production while actively suppressing the Dicer-dependent processing of other microRNAs, thereby ensuring an efficient and precise mechanism to control terminal erythrocyte differentiation. Recent work from our lab and others indicate that miR-451 comprises up to 60% of the microRNA content of maturing erythrocytes, while miR-144 that is co-expressed as a cluster with miR-451 and processed by Dicer only accounts for 1.5%. These results are in striking contrast with our preliminary data that shows that Ago2- dependent processing is not as efficient as Dicer-mediated biogenesis. Surprisingly, our most recent data suggests that pre-miR-451 represses canonical microRNA biogenesis and that Dicer is a target of miR-144. Leveraging all these data, the current proposal examines the hypothesis that miR-451 has a second activity, unrelated to its sequence but dependent on its structural features as a competitive inhibitor of Dicer. Three highly integrated but not interdependent Aims will address the above hypothesis using a set of reprogrammed pre-miR-451 hairpins to i) determine the biochemical features of miR-451/Dicer interaction and their role on canonical miRNA biogenesis, ii) uncover the role of miR-144 in miR-451 processing and iii) identify the erythropoietic processes that most depend on the Ago2-mediated biogenesis of miR-451. Using zebrafish and human iPSC as a model system, the current proposal uses novel genetic and molecular approaches to mechanistically probe the interplay of canonical and non-canonical microRNA processing pathways during erythropoiesis. In doing so, it will uncover mechanisms with the potential to instruct future improvements in blood production form iPSC and therapeutic interventions on anemia. The novelty of the proposal is also driven by an interdisciplinary team that combines experience in microRNAs, zebrafish, bioinformatics and iPSC differentiation. The successful completion of this project will transform our understanding of how microRNAs regulate cell fate and provide invaluable insights to improve iPSC reprogramming to erythrocytes.

项目总结：“microRNA的非典型功能分析” 对细胞命运决定的承诺是正确的胚胎发育和组织稳态的基础。 microRNA是一个小的非编码RNA家族，是积极参与许多细胞凋亡的因素之一。 分化过程。事实上，在红细胞生成过程中分化的最后一步部分是由miR-21调控的。 451和它的死亡导致严重贫血。令人惊讶的是，miR-451是唯一已知的微RNA， 是Dicer独立的但Ago 2依赖的。奇怪的是，Dicer仍然在红细胞中表达，从而提高了红细胞中Dicer的表达。 问题是红细胞生成通过非典型的 通路该项目的目标是揭示miR-451的非经典加工机制， 成为红细胞分化不可或缺的条件。我们假设这种替代加工途径 有利于miR-451的产生，同时积极抑制其他microRNA的Dicer依赖性加工， 从而确保控制终末红细胞分化的有效和精确的机制。 我们实验室和其他实验室的最新工作表明，miR-451包含高达60%的microRNA含量， 成熟红细胞，而与miR-451共表达为簇并由Dicer加工的miR-144 只占百分之一点五。这些结果与我们的初步数据形成鲜明对比，这些数据表明Ago 2- 依赖性加工不如Dicer介导的生物发生有效。令人惊讶的是，我们最新的数据 提示pre-miR-451抑制典型microRNA生物发生，Dicer是miR-144的靶点。 利用所有这些数据，目前的提议检验了miR-451具有第二活性的假设， 与其序列无关，但依赖于其作为Dicer竞争性抑制剂的结构特征。 三个高度综合但不相互依赖的目标将使用一套 重编程的前-miR-451发夹以i）确定miR-451/Dicer相互作用的生物化学特征，和 它们在典型miRNA生物发生中的作用，ii）揭示miR-144在miR-451加工中的作用，iii）鉴定 最依赖于Ago 2介导的miR-451生物合成的红细胞生成过程。利用斑马鱼 和人类iPSC作为模型系统，目前的建议使用新的遗传和分子方法， 机械地探测典型和非典型microRNA加工途径的相互作用， 红细胞生成在这样做的过程中，它将揭示具有指导未来血液改善的潜力的机制。 生产形式iPSC和贫血的治疗干预。该提案的新奇也受到一个 跨学科团队，结合了microRNA、斑马鱼、生物信息学和iPSC方面的经验 分化该项目的成功完成将改变我们对microRNA如何 调节细胞命运，并提供宝贵的见解，以改善iPSC重编程为红细胞。