Analysis of non-canonical functions of microRNAs

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常胚胎发育和组织的基础 体内平衡microRNA是一个小的非编码RNA家族，是积极参与 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部分是 由miR-451控制，其死亡导致严重贫血。令人惊讶的是，miR-451是唯一已知的 其加工不依赖于Dicer但依赖于Ago 2。奇怪的是， 在红细胞中表达，这就提出了一个问题： 通过非经典途径处理miR-451。这个项目的目标是揭示 机制，miR-451的非典型加工成为红细胞不可或缺的 分化我们假设这种替代加工途径有利于miR-451的产生， 积极抑制其他microRNA的Dicer依赖性加工，从而确保有效和 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的最新工作表明，miR-451占microRNA含量的60%。 而miR-144与miR-451共表达为簇，并被成熟红细胞加工， 切丁只占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， Ago 2依赖的加工不如Dicer介导的生物合成有效。令人惊讶的是，我们最近的 数据表明，pre-miR-451抑制典型的microRNA生物合成，Dicer是miR-451的靶点。 144.利用所有这些数据，目前的提案检验了miR-451具有第二个 活性，与其序列无关，但取决于其作为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 以及它们在典型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生物发生的红细胞生成过程。 使用斑马鱼和人类iPSC作为模型系统，目前的提议使用新的遗传和分子生物学方法， 机械探测典型和非典型microRNA加工相互作用的方法 红细胞生成过程中的通路。在这样做的过程中，它将揭示具有指导未来的潜力的机制。 iPSC造血功能的改善和对贫血的治疗干预。的新奇 该提案也是由一个跨学科团队推动的，该团队将microRNA，斑马鱼， 生物信息学和iPSC分化。该项目的成功完成将改变我们的 了解microRNA如何调节细胞命运，并为改善iPSC提供宝贵的见解 重编程为红细胞。

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.