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

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参与了许多不同的步骤，最终决定了细胞内RNA的命运。随着在突触中发现microRNA（miRNAs）及其在神经元RNA颗粒中的存在，人们很快就清楚了非编码RNA（ncRNA）是脑中基因表达的重要转录后调节因子，在脑中它们对神经发生和突触可塑性做出了重要贡献。在这里，反式作用因子，miRNA和RBP之间的动态相互作用，通过其3-UTR的调节靶转录物，使细胞能够严格调节的mRNA.In本项目的第二个资金周期，我们的目标是表征RBP在调节miRNA与mRNA靶相互作用的作用。在这里，我们将重点放在脑特异性双链RBP Staufen 2（Stau 2），有助于树突状mRNA定位和随后的局部蛋白质合成在激活的突触。为了从机制和功能上深入了解miRNA和RBP在靶mRNA的3-UTR处的迷人相互作用，我们选择了两个高置信度的Stau 2靶点，即小G蛋白信号传导调节因子（Rgs 4）mRNA和钙调蛋白3（Calm 3）mRNA。目的1：进一步分析Stau 2对miR-26 a/Rgs 4 mRNA的动态调控，以确定Stau 2和miR-26 a是否以拮抗的方式调控Rgs 4 3-UTR。此外，我们将研究位于Calm 3中保留内含子内的预测的miR-137结合位点是否确实具有功能并用于调节Calm 3转录。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Mov 10，Ago 2和PACT以及Stau 2。在这里，我们将利用实验室建立的MS 2 RNA成像系统，在培养的原代神经元中研究上述组分组装/拆卸成miRNP的动力学。最后，在长期目标3中，我们将通过研究它们在WT以及在前脑神经元中Stau 2缺陷的转基因大鼠中的功能贡献来研究miRNA/mRNA/Stau 2相互作用的体内生理功能。总之，我们预计我们的研究结果将揭示如何参与树突mRNA定位和突触翻译的RBP通过调节ncRNA与其生理靶mRNA的相互作用来严格控制突触功能。这也将推进我们对突触在学习过程中如何经历经验依赖性修改的理解。我们相信，这些知识的获得不仅对了解正常的大脑功能很重要，而且对了解神经系统疾病的各种功能障碍也很重要。

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.