Molecular mechanisms and functional consequences of miR-128 regulation in cortical development

从神经发生和细胞命运选择的控制到突触发生和功能连接的建立，在确定单个mirna对中枢神经系统形态发生的许多步骤的贡献方面已经取得了重大进展。我们一直在研究神经系统中最丰富的mirna之一miR-128及其在神经元迁移、树突分支和内在兴奋性中的发育作用。哺乳动物有两个miR-128拷贝，每个拷贝都位于同源蛋白R3HDM1（用于miR-128-1）和ARPP21（用于主要的脑同种异构体miR-128-2）的两个基因之一的内含子内。R3HDM1和ARPP21编码保守的、假定的rna结合蛋白，其特征是R3H和SUZ结构域。我们通过个体核苷酸分辨率交联和免疫沉淀（iCLIP）来描述ARPP21的rna结合活性。ARPP21与富尿嘧啶序列基序结合具有高特异性，对转录本的3 ' UTR具有强烈的偏好。ARPP21- rna与一组经过验证的靶mrna相互作用的功能表征表明，ARPP21是基因表达的转录后激活因子。ARPP21结合的生物信息学分析显示，ARPP21和miR-128的下游靶点显著重叠，并且在KEGG通路mRNA监测、TGFß和神经营养因子信号通路中显著富集。这导致了一个令人惊讶的模型，即一个基因产生具有共同靶标的转录后抑制因子（miR-128）和激活因子（ARPP21）。我们已经证实了UPF1、CASC3、MSI2 （mRNA监视）、MSK1、CREB1（神经营养因子信号传导）和PHF6（树突状生长）mRNA的拮抗共调节。miR-128与ARPP21的分子拮抗作用还体现在功能水平上。在体内操纵miR-128和ARPP21的表达，我们发现敲低ARPP21模仿miR-128过表达表型降低树突复杂性，而异位ARPP21表达导致树突复杂性增加。在此基础上，我们提出了一项研究计划，以解决这一新的调控途径的基本分子和细胞特性以及发育相关性。我们将通过体内iCLIP和核糖体分析的结合来表征神经元培养物和大脑中ARPP21 rna结合的生理库。我们还将使用CRISPR-Cas9介导的miRNA基因编辑来探索ARPP21与其他神经元miRNA之间的功能相互作用。类似地，我们的iCLIP图谱使我们能够识别ARPP21的下游效应物，包括几种rna结合蛋白。这将为我们研究神经元中ARPP21的细胞内动力学和mirna介导的转录后调控提供信息。最后，我们将分析如何调节ARPP21和miR-128之间的平衡，以及该系统如何指定树突分支并影响神经元的兴奋性。

Great strides have been made in defining the contribution of individual miRNAs for each of the many steps in CNS morphogenesis, from the control of neurogenesis and cell fate choice to synaptogenesis and the establishment of functional connectivity. We have been studying miR-128, one of the most abundant miRNAs in the nervous system, and its developmental roles in neuronal migration, dendritic branching and intrinsic excitability. Mammals have two copies of miR-128, each invariably located within an intron of one of two genes for the homologous proteins R3HDM1 (for miR-128-1) and ARPP21 (for the major brain isoform miR-128-2). R3HDM1 and ARPP21 encode conserved, putative RNA-binding proteins characterized by R3H and SUZ domains. We performed individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to describe the RNA-binding activity of ARPP21. ARPP21 binds with high specificity to a uridine-rich sequence motif with a strong preference for the 3-prime UTR of transcripts. Functional characterization of ARPP21-RNA interactions for a panel of validated target mRNAs revealed that ARPP21 acts as a post-transcriptional activator of gene expression. Bioinformatic analysis of ARPP21 binding revealed that downstream targets of ARPP21 and miR-128 significantly overlap and are significantly enriched for the KEGG pathways mRNA surveillance, and the TGFß and neurotrophin signalling pathways. This leads to the surprising model that one gene produces a post-transcriptional repressor (miR-128) and activator (ARPP21) that share common targets. We have verified this antagonistic co-regulation of mRNAs for UPF1, CASC3, MSI2 (mRNA surveillance), MSK1, CREB1 (neurotrophin signaling) and PHF6 (dendritic outgrowth). The molecular antagonism between miR-128 and ARPP21 is also reflected at the functional level. Manipulating the expression of miR-128 and ARPP21 in vivo, we found that knockdown of ARPP21 mimics the miR-128 overexpression phenotype of reduced dendritic complexity whereas ectopic ARPP21 expression leads to an increase in dendritic complexity.Based on this work, we propose a research program to address the fundamental molecular and cellular properties and the developmental relevance of this new regulatory pathway. We will characterize the physiological repertoire of ARPP21 RNA-binding in neuronal cultures and the brain by a combination of in vivo iCLIP and ribosomal profiling. We will also use CRISPR-Cas9 mediated miRNA gene editing to probe the functional interactions between ARPP21 and other neuronal miRNAs. Similarly, our iCLIP profile allows us to identify downstream effectors of ARPP21, including several RNA-binding proteins. This will inform our efforts to study the intracellular dynamics of ARPP21 and miRNA-mediated post-transcriptional regulation in neurons. Finally, we will analyze how the balance between ARPP21 and miR-128 is regulated, and how the system specifies dendritic branching and influences neuronal excitability.