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

皮质发育中 miR-128 调节的分子机制和功能后果

• 批准号: 255046332
• 负责人: Dr. F. Gregory Wulczyn
• 金额: --
• 依托单位: Institut für Zell- und Neurobiologie (CCM)
• 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/255046332?language=en
• 关键词: Molecular; mechanisms; functional; consequences; miR

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.

在确定单个miRNA对CNS形态发生的许多步骤中的每一个步骤的贡献方面已经取得了很大的进展，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。我们一直在研究miR-128，神经系统中最丰富的miRNAs之一，及其在神经元迁移，树突分支和内在兴奋性中的发育作用。哺乳动物有两个拷贝的miR-128，每个拷贝总是位于同源蛋白R3 HDM 1（对于miR-128-1）和ARPP 21（对于主要的脑同种型miR-128-2）的两个基因之一的内含子内。R3 HDM 1和ARPP 21编码保守的、推定的RNA结合蛋白，其特征在于R3 H和SUZ结构域。我们进行了单个核苷酸解析交联和免疫沉淀（iCLIP）来描述ARPP 21的RNA结合活性。ARPP 21以高特异性结合富含尿苷的序列基序，对转录物的3-prime UTR具有强烈偏好。一组经验证的靶mRNA的ARPP 21-RNA相互作用的功能表征显示，ARPP 21充当基因表达的转录后激活剂。ARPP 21结合的生物信息学分析显示，ARPP 21和miR-128的下游靶点显著重叠，并且显著富集KEGG途径mRNA监视以及TGF β和神经营养因子信号传导途径。这导致了一个令人惊讶的模型，即一个基因产生一个转录后阻遏物（miR-128）和激活物（ARPP 21），它们具有共同的靶点。我们已经验证了UPF 1、CASC 3、MSI 2（mRNA监视）、MSK 1、CREB 1（神经营养因子信号传导）和PHF 6（树突状生长）的mRNA的这种拮抗性共调节。miR-128与ARPP 21之间的分子拮抗作用也反映在功能水平上。通过在体内调控miR-128和ARPP 21的表达，我们发现ARPP 21的敲低可以模拟miR-128过表达降低树突复杂性的表型，而ARPP 21的异位表达可以导致树突复杂性的增加。我们将通过结合体内iCLIP和核糖体分析来表征神经元培养物和大脑中ARPP 21 RNA结合的生理库。我们还将使用CRISPR-Cas9介导的miRNA基因编辑来探测ARPP 21和其他神经元miRNA之间的功能相互作用。同样，我们的iCLIP图谱使我们能够识别ARPP 21的下游效应子，包括几种RNA结合蛋白。这将为我们研究ARPP 21的细胞内动力学和miRNA介导的神经元转录后调控提供信息。最后，我们将分析ARPP 21和miR-128之间的平衡是如何调节的，以及该系统如何指定树突分支并影响神经元的兴奋性。