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

在确定单个miRNAs在中枢神经系统形态发生的许多步骤中的作用方面取得了很大进展，从控制神经发生和细胞命运选择到突触发生和功能连接的建立。我们一直在研究miR-128，神经系统中含量最丰富的miRNAs之一，以及它在神经元迁移、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(miR-128-1)和ARPP21(大脑主要亚型miR-128-2)的两个基因中的一个内含子内。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。我们进行了单核苷酸拆分、交联和免疫沉淀(ICLIP)来描述ARPP21的RNA结合活性。ARPP21与一个富含尿苷的序列基序具有高度的特异性，并对转录本的3个碱基UTR有强烈的偏好。对一组有效的靶mRNAs的ARPP21-RNA相互作用的功能鉴定表明，ARPP21作为基因表达的转录后激活因子。对ARPP21结合的生物信息学分析表明，ARPP21和miR-128的下游靶点显著重叠，并显著丰富了KEGG通路的mRNA监测，以及转化生长因子和神经营养因子信号通路。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抑制因子(miR-128)和激活因子(ARPP21)，它们共享共同的靶标。我们已经证实了这种对UPF1、CASC3、MSI2(mRNA监视)、MSK1、CREB1(神经营养因子信号)和PHF6(树突状突起)的mRNAs的拮抗协同调节。MiR-128和ARPP21之间的分子拮抗作用也反映在功能水平上。在体内操纵miR-128和ARPP21的表达，我们发现ARPP21的敲除模仿了miR-128过表达的表型，降低了树突状细胞的复杂性，而异位表达的ARPP21导致树突状细胞复杂性的增加。在此基础上，我们提出了一个研究计划，以解决这一新的调控途径的基本分子和细胞特性以及与发育的相关性。我们将通过体内iCLIP和核糖体分析相结合的方法来表征神经元培养和大脑中ARPP21RNA结合的生理图谱。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miRNAs之间的功能相互作用。同样，我们的iCLIP图谱使我们能够识别ARPP21的下游效应器，包括几个RNA结合蛋白。这将有助于我们努力研究ARPP21的细胞内动力学和miRNA介导的神经元转录后调控。最后，我们将分析ARPP21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和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.