Circular RNAs: novel regulators of dendritic protein synthesis during mammalian synapse development

环状RNA：哺乳动物突触发育过程中树突蛋白合成的新型调节因子

• 批准号: 255069996
• 负责人: Professor Dr. Christoph Dieterich
• 金额: --
• 依托单位: Klinik für Kardiologie, Angiologie und Pulmologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/255069996?language=en
• 关键词: Circular; RNAs; novel; regulators; dendritic

The precise regulation of local protein synthesis in neuronal dendrites is essential for activity-dependent synapse formation, remodeling and plasticity, and defects in this process are linked to neurodevelopmental disorders, including mental retardation and autism. Research during the last decade has identified microRNAs (miRNAs), an extensive class of non-coding RNAs, and RNA-binding proteins (RBP) as important regulators of local protein synthesis and dendrite morphogenesis in neurons. However, how miRNA/RBP function itself is orchestrated by neuronal activity is largely uknown. During the first funding period, we focused the role of a miRNA/RBP interplay in the regulation of dendrite morphology. Thereby, we identified two RBPs, Nova1 and Ncoa3, that are required for the repressive activity of dendritic miRNAs. Whereas Nova1 is part of neuronal miRISC, Ncoa3 indirectly stimulates miRISC function by inducing Ago2 expression (Störchel et al., EMBO J. 2015). We further identified a competing endogenous RNA, Ube3a-1, as an additional non-coding regulatory mechanism that controls the availability of specific dendritic miRNAs (Valluy et al., Nat. Neurosci 2015). In the second funding period, we plan to focus on circular RNAs (circRNAs), a subclass of long non-coding RNAs that are generated by specific head-to-tail splicing events (backsplicing). In preliminary experiments, we found that circRNAs in rat hippocampal neurons were abundant, highly enriched in the dendritic compartment and regulated by neuronal activity. RNAi-mediated knockdown of specific circRNAs impaired BDNF-dependent dendritogenesis, suggesting their involvement in neural circuit development. In silico analysis indicated the presence of translational start sites and RBP binding motifs in specific dendritic circRNAs, providing a rationale for further mechanistic studies. Together, this led us to hypothesize that circRNAs are important regulators of dendritic protein synthesis and activity-dependent synapse development. To address this hypothesis, we will pursue the following specific aims: (1) to comprehensively validate dendritic localization of candidate circRNAs identified by RNAseq in vitro and in vivo; (2) to interrogate the function of up to 24 candidate circRNAs in activity-dependent synapse development and plasticity of hippocampal neurons using RNAi; (3) to unravel the internal sequence and structure of dendritic circRNAs by long RNA-seq approaches; (4) to identify the mechanism underlying dendritic circRNA function, with a focus on miRNA/RBP sponges, dendrite delivery vehicles and mRNA traps. By addressing these aims, we will obtain unprecedented insight into the role of circRNAs in mammalian synapse development and plasticity. This will provide a framework for future studies that will address circRNA function in a more physiological context in the intact brain.

神经元树突中局部蛋白质合成的精确调节对于活性依赖性突触形成、重塑和可塑性至关重要，并且该过程中的缺陷与神经发育障碍（包括智力迟钝和自闭症）有关。在过去的十年中，研究已经确定microRNAs（miRNAs），一个广泛的类别的非编码RNA，和RNA结合蛋白（RBP）作为重要的调节剂的局部蛋白质合成和树突形态发生的神经元。然而，miRNA/RBP功能本身是如何通过神经元活动来协调的在很大程度上是未知的。在第一个资助期间，我们专注于miRNA/RBP相互作用在树突形态调控中的作用。因此，我们确定了两个RBP，Nova 1和Ncoa 3，这是树突状miRNA的抑制活性所必需的。而Nova 1是神经元miRISC的一部分，Ncoa 3通过诱导Ago 2表达间接刺激miRISC功能（Störchel等人，EMBO J. 2015）。我们进一步鉴定了竞争性内源RNA Ube 3a-1作为控制特定树突状miRNA的可用性的额外非编码调节机制（Valluy等人，Nat. Neurosci 2015）。在第二个资助期内，我们计划专注于环状RNA（circRNA），这是一种由特定的头到尾剪接事件（反向剪接）产生的长非编码RNA亚类。在初步实验中，我们发现大鼠海马神经元中的circRNA是丰富的，高度富集在树突区室中，并受神经元活动的调节。RNAi介导的特定circRNA的敲低损害了BDNF依赖的树突状细胞发生，表明它们参与了神经回路的发育。计算机模拟分析表明在特定的树突状circRNA中存在翻译起始位点和RBP结合基序，为进一步的机制研究提供了理论基础。总之，这使我们假设circRNA是树突蛋白合成和活性依赖性突触发育的重要调节因子。为了解决这一假设，我们将追求以下具体目标：（1）在体外和体内全面验证通过RNAseq鉴定的候选circRNA的树突定位;（2）使用RNAi询问多达24个候选circRNA在海马神经元的活动依赖性突触发育和可塑性中的功能;（3）通过长RNA测序方法揭示树突状circRNA的内部序列和结构;（4）确定树突状circRNA功能的潜在机制，重点是miRNA/RBP海绵，树突状递送载体和mRNA陷阱。 通过解决这些目标，我们将获得前所未有的洞察力，了解circRNA在哺乳动物突触发育和可塑性中的作用。这将为未来的研究提供一个框架，这些研究将在完整大脑中更生理的背景下解决circRNA功能。