Dissecting the mechanisms of miRNA function in establishment of neuronal polarity

神经元是由细胞体或索马和细胞延伸或神经突形成的高度极化的细胞。这种极性对神经元的正常功能至关重要。细胞极性的建立主要依赖于mRNA的不对称亚细胞定位和翻译。在我们之前的工作中，我们已经开发了神经突/索马体分离方案，结合质谱、RNA-seq、Ribo-seq和生物信息学分析，以鉴定在神经元细胞的神经突和索马体之间差异定位和翻译的蛋白质和RNA。此外，我们发现mRNA定位和局部翻译受到microRNA（miRNA）途径废除的强烈影响。miRNAs是基因表达的重要调节因子，在翻译抑制和mRNA降解中具有已知的作用，靶向超过一半的真核基因。然而，它们在mRNA的亚细胞定位和翻译中的功能尚未得到系统的分析。在拟议的研究中，我们的目标是了解网络的miRNA介导的调节mRNA的定位和翻译模式在神经元细胞。我们将使用AGO PAR-CLIP定位和局部翻译的mRNA中的miRNA结合位点，进行综合计算数据分析以鉴定调节亚细胞定位和翻译模式的miRNA，并剖析潜在的作用机制。这项研究有可能为细胞极性建立的机制和miRNAs在这一过程中的作用提供重要的见解。

Neuron is a highly polarized cell formed by a cell body, or soma, and cell extensions, or neurites. Such polarity is crucial to proper neuronal function. Establishment of cell polarity in large relies on asymmetric subcellular localization and translation of mRNAs. In our prior work, we have developed neurite/soma fractionation scheme in combination with mass spectrometry, RNA-seq, Ribo-seq and bioinformatics analyses, to identify proteins and RNAs that are differentially localized and translated between neurites and soma of neuronal cells. Moreover, we showed that mRNA localization and local translation are strongly affected by abrogation of the microRNA (miRNA) pathway. miRNAs are important regulators of gene expression with known roles in translational repression and mRNA decay, targeting more than a half of all eukaryotic genes. However their functions in subcellular localization and translation of mRNAs have not been systematically analyzed. In the proposed study, we aim to understand the network of miRNA-mediated regulation of mRNA localization and translation patterns in neuronal cells. We will map miRNA-binding sites in localized and locally translated mRNAs using AGO PAR-CLIP, perform integrative computational data analysis to identify miRNAs that regulate subcellular localization and translation patterns and dissect the underlying mechanisms of action. The proposed research has the potential to provide important insights into the mechanisms underlying establishment of cell polarity and roles of miRNAs in this process.