RNA-binding proteins as regulators of non coding RNA function at the synapse

RNA 结合蛋白作为突触非编码 RNA 功能的调节剂

• 批准号: 254897163
• 负责人: Professor Dr. Michael Kiebler
• 金额: --
• 依托单位: Lehrstuhl Anatomie III - Zellbiologie
• 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 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/254897163?language=en
• 关键词: RNA; binding; proteins; regulators; non

RNA-binding proteins (RBPs) regulate key cellular functions, e.g. nuclear RNA processing and modification, RNA export and cytoplasmic localization, translation and degradation. In neurons, RBPs are involved in many distinct steps that ultimately decide on the fate of RNAs within the cell. With the discovery of microRNAs (miRNAs) at synapses and their presence in neuronal RNA granules, it became quickly clear that non-coding RNAs (ncRNA) are important post-transcriptional regulators of gene expression in the brain, where they critically contribute to neurogenesis and synaptic plasticity. Here, the dynamic interplay between trans-acting factors, miRNAs and RBPs, critically contributes to the regulation of target transcripts via their 3-UTRs, enabling the cell to tightly regulate the fate of the mRNA.In the second funding period of this project, we aim to characterize the role of RBPs in regulating miRNA interactions with their mRNA targets. Here we would focus on the brain-specific double-stranded RBP Staufen2 (Stau2) that contributes to dendritic mRNA localization and subsequent local protein synthesis at the activated synapse. To get mechanistic and functional insight into the fascinating interplay between miRNAs and RBPs at the 3-UTR of target mRNAs, we have chosen two high confidence Stau2 targets, i.e. the regulator of small G protein signaling (Rgs4) mRNA and the calmodulin3 (Calm3) mRNA. In aim 1, we will further analyze the dynamic regulation of miR26a/Rgs4 mRNA by Stau2, to determine whether Stau2 and miR-26a regulate Rgs4 3-UTR in an antagonistic fashion. In addition, we will investigate whether the predicted miR-137 binding site located within the retained intron in Calm3 is indeed functional and is used to regulate the Calm3 transcript. Next (aim 2), we aim at investigating whether and how synaptic activity modulates this interaction and how it affects the association and localization of RISC factors, e.g. Mov10, Ago2 and PACT, together with Stau2. Here, we will take advantage of the MS2 RNA imaging system established in the lab to study in cultured primary neurons the assembly/disassembly dynamics of the aforementioned components into miRNPs. Finally, in the long-term aim 3 we will investigate the in vivo physiological function of the miRNA/mRNA/Stau2 interplay by studying their functional contributions both in WT as well as in transgenic rats that are Stau2-deficient in forebrain neurons. In summary, we anticipate that our results will shed new light on how RBPs involved in dendritic mRNA localization and synaptic translation critically control synaptic function by regulating the interaction of ncRNAs with their physiological target mRNAs. This will also advance our understanding of how synapses undergo experience-dependent modifications during learning. We are convinced that this gain of knowledge will not only be important to understand normal brain function, but also various dysfunctions underlying neurological diseases.

RNA 结合蛋白 (RBP) 调节关键的细胞功能，例如核RNA加工和修饰、RNA输出和细胞质定位、翻译和降解。在神经元中，RBP 参与许多不同的步骤，最终决定细胞内 RNA 的命运。随着突触中 microRNA (miRNA) 的发现及其在神经元 RNA 颗粒中的存在，人们很快就清楚非编码 RNA (ncRNA) 是大脑中基因表达的重要转录后调节因子，它们对神经发生和突触可塑性发挥着至关重要的作用。在这里，反式作用因子、miRNA 和 RBP 之间的动态相互作用，对通过 3-UTR 调节靶标转录物至关重要，使细胞能够严格调节 mRNA 的命运。在该项目的第二个资助期，我们的目标是表征 RBP 在调节 miRNA 与其 mRNA 靶标相互作用中的作用。在这里，我们将重点关注大脑特异性双链 RBP Staufen2 (Stau2)，它有助于树突 mRNA 定位以及随后在激活的突触处的局部蛋白质合成。为了深入了解靶 mRNA 3-UTR 处 miRNA 和 RBP 之间令人着迷的相互作用，我们选择了两个高置信度的 Stau2 靶标，即小 G 蛋白信号传导 (Rgs4) mRNA 的调节器和钙调蛋白 3 (Calm3) mRNA。在目标1中，我们将进一步分析Stau2对miR26a/Rgs4 mRNA的动态调节，以确定Stau2和miR-26a是否以拮抗方式调节Rgs4 3-UTR。此外，我们将研究位于 Calm3 保留内含子内的预测 miR-137 结合位点是否确实具有功能并用于调节 Calm3 转录本。接下来（目标 2），我们的目标是研究突触活动是否以及如何调节这种相互作用，以及它如何影响 RISC 因子的关联和定位，例如Mov10、Ago2 和 PACT，以及 Stau2。在这里，我们将利用实验室建立的MS2 RNA成像系统，在培养的原代神经元中研究上述成分在miRNP中的组装/分解动态。最后，在长期目标 3 中，我们将通过研究 miRNA/mRNA/Stau2 相互作用在 WT 以及前脑神经元中 Stau2 缺陷的转基因大鼠中的功能贡献来研究 miRNA/mRNA/Stau2 相互作用的体内生理功能。总之，我们预计我们的结果将为参与树突 mRNA 定位和突触翻译的 RBP 如何通过调节 ncRNA 与其生理目标 mRNA 的相互作用来关键控制突触功能提供新的线索。这也将增进我们对突触在学习过程中如何经历依赖于经验的修改的理解。我们相信，这种知识的获取不仅对于理解正常的大脑功能很重要，而且对于理解神经系统疾病背后的各种功能障碍也很重要。