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

点击翻译按钮获取中文摘要

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.