Analysis of non-canonical functions of microRNAs

项目摘要：“微型核糖核酸的非规范功能分析” 对细胞命运决定的承诺是正确的胚胎发育和组织的基础 动态平衡。MicroRNAs是一个小的非编码RNA家族，是积极参与的因素之一 在许多分化过程中。事实上，红细胞生成过程中分化的最后一步是 由miR-451控制，它的死亡会导致严重的贫血。令人惊讶的是，miR-451是唯一已知的 其处理过程不依赖于Dever但依赖于Ago2的microRNA。矛盾的是，迪格尔仍然 在红细胞中表达，这提出了一个问题，即对红细胞生成有什么可能的好处 通过非规范途径处理miR-451。这个项目的目标是通过 MiR-451的非正则加工成为红细胞不可缺少的机制 差异化。我们假设，这种替代的加工途径有利于miR-451的生产，而 积极抑制其他microRNA的依赖于Dice的加工，从而确保高效和 控制红细胞终末分化的精确机制。 我们实验室和其他实验室最近的工作表明，miR-451包含高达60%的microRNA含量 而miR-144与miR-451以簇的形式共同表达，并经 迪格尔仅占1.5%。这些结果与我们的初步数据形成鲜明对比，初步数据显示 依赖于AGO2的加工不如迪格尔介导的生物发生那么有效。令人惊讶的是，我们最新的 数据表明，前miR-451抑制了典型的microRNA的生物发生，而Disher是miR-451的靶标。 144.利用所有这些数据，目前的提议检验了miR-451有第二个 活性，与其序列无关，但依赖于其结构特征，作为DICER的竞争性抑制物。 三个高度集成但不相互依赖的目标将使用一组 重新编程的预miR-451发夹以I)确定miR-451/DICER相互作用的生化特征 以及它们在规范的miRNA生物发生中的作用，II)揭示了miR-144在miR-451加工和 Iii)确定最依赖于Ago2介导的miR-451生物发生的红细胞生成过程。 以斑马鱼和人类IPSC为模型系统，目前的方案使用了新的遗传和分子 从机械上探索规范和非规范microRNA加工的相互作用的方法 红血球生成过程中的途径。在这样做的过程中，它将发现有可能指导未来的机制 IPSC和贫血治疗干预措施对血液产量的改善。它的新奇之处在于 该提案还由一个跨学科团队推动，该团队结合了microRNA、斑马鱼、 生物信息学和IPSC差异化。这个项目的成功完成将改变我们的 了解microRNAs如何调控细胞命运并为改善IPSC提供宝贵的见解 对红血球进行重新编程。

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.