Identification and functional analysis of Cdc42 zipcodes

RNA的适当亚细胞定位和局部翻译调节在高度区室化的细胞（如神经元）中至关重要。RNA定位由通常在mRNA 3 'UTR中发现的特异性顺式调节元件介导。因此，产生选择性3 'UTR的过程-选择性剪接和聚腺苷酸化-具有使神经元中的mRNA定位模式多样化的潜力。我们已经对从mESC衍生的神经元分离的神经突和索马中的替代3 'UTR进行了定位1。我们的分析确定了593个基因的差异定位的3 'UTR亚型。特别是，我们已经表明，两个异构体的Cdc 42基因与不同的功能，在神经元极性的差异定位之间的突起和索马的mESC衍生的和小鼠原代皮层神经元，在mRNA和蛋白质水平。使用报告基因分析和3 'UTR交换实验，我们已经确定了替代性3' UTR和mRNA转运在替代性CDC 42蛋白亚型的差异定位中的作用。此外，我们使用SILAC来鉴定在Cdc 42定位和翻译中具有潜在作用的同种型特异性Cdc 42 3 'UTR结合蛋白质组。我们的分析指出使用替代3 'UTR同种型作为一种新的机制，以提供功能多样的替代蛋白质同种型的差异定位。在目前的建议中，我们将通过识别Cdc 42邮政编码及其结合的RBP来剖析介导Cdc 42亚型差异定位的机制，并分析介导不同功能的Cdc 42蛋白亚型的机制。

The proper subcellular localization of RNAs and local translational regulation is crucial in highly compartmentalized cells, such as neurons. RNA localization is mediated by specific cis-regulatory elements usually found in mRNA 3'UTRs. Therefore, processes that generate alternative 3'UTRs – alternative splicing and polyadenylation – have the potential to diversify mRNA localization patterns in neurons. We have performed mapping of alternative 3'UTRs in neurites and soma isolated from mESC-derived neurons1. Our analysis identified 593 genes with differentially localized 3'UTR isoforms. In particular, we have shown that two isoforms of Cdc42 gene with distinct functions in neuronal polarity are differentially localized between neurites and soma of mESC-derived and mouse primary cortical neurons, at both mRNA and protein level. Using reporter assays and 3'UTR swapping experiments, we have identified the role of alternative 3’UTRs and mRNA transport in differential localization of alternative CDC42 protein isoforms. Moreover, we used SILAC to identify isoform-specific Cdc42 3'UTR-bound proteome with potential role in Cdc42 localization and translation. Our analysis points to usage of alternative 3'UTR isoforms as a novel mechanism to provide for differential localization of functionally diverse alternative protein isoforms. Within the current proposal, we will dissect the mechanisms mediating differential localization of Cdc42 isoforms, by identifying the Cdc42 zipcodes and their bound RBPs, and analyze the mechanisms mediating different functionality of CDC42 protein isoforms.