Pumilio2-mediated control of local protein expression in neurons

突触局部蛋白质合成的调节在初级神经元中尤为重要。在这里，一组特定的mRNAs在RNA结合蛋白(RBP)的帮助下被包装成核糖核蛋白颗粒(RNP或RNA颗粒)，并沿着微管运输到突触附近的树突。人们普遍认为，翻译在运输过程中受到抑制。然而，涉及翻译调控的潜在机制和分子仍然知之甚少。神经元特异性RBP Pumilio2(Pum2)在许多生物体和细胞类型中是一种众所周知的、保守的翻译调节因子。在这里，Pum2参与了eIF4E mRNA的翻译调控以及编码几个钠通道的mRNAs，如Nav1.1和Nav1.6。Pum2缺陷的原代海马神经元表现出树突棘形态发生和神经元兴奋性的缺陷。值得注意的是，Pum2基因在小鼠体内的敲除会导致癫痫发作。Pum2是含有Staufen2(Stau2)的RNPs的一个成分，Stau2是另一个参与树突状mRNA定位的众所周知的RBP。因此，很容易推测Pum2参与了神经元局部转录表达的控制。为了系统地了解Pum2在这一过程中的作用，我们计划将从小鼠脑中提取RNA颗粒与野生型和Pum2缺陷小鼠脑的多聚体梯度分离相结合，以确定生理上相关的Pum2靶标mRNAs。在与Medenbach(项目9)的合作中，我们将建立一个从小鼠神经元组织到概括调控的体外翻译系统。与Kiebler实验室复杂的小鼠模型相结合，我们将能够阐明Pum2介导的蛋白质合成控制的潜在机制，特别是在成熟突触的局部翻译，并获得对错误调控如何导致疾病的分子洞察力。

Regulation of local protein synthesis at the synapse is particularly important in primary neurons. Here, a set of specific mRNAs is packaged with the help of RNA-binding proteins (RBPs) into ribonucleoprotein particles (RNPs or RNA granules) and transported along microtubules into dendrites near synapses. It is widely believed that translation is repressed during transport. However, the underlying mechanisms and molecules involved in translational regulation remain poorly understood. The neuron-specific RBP Pumilio2 (Pum2) is a well-known, conserved translational regulator in many organisms and cell types. Here, Pum2 has been implicated in the translational regulation of eIF4E mRNA as well as mRNAs coding for several sodium channels such as Nav1.1 and Nav1.6. Pum2-deficient primary hippocampal neurons display deficits in dendritic spine morphogenesis as well as in the excitability of neurons. It is important to note that Pum2 knock-down in mice causes epileptic seizures. Pum2 is a component of Staufen2 (Stau2)-containing RNPs, with Stau2 being another well-known RBP involved in dendritic mRNA localization. Therefore, it is tempting to speculate that Pum2 is involved in the control of local expression of localized transcripts in neurons. To gain systematic insight into the role of Pum2 in this process, we plan to combine RNA granule isolation from mouse brain with polysome gradient fractionation of wild type and Pum2-deficient mouse brains to identify physiologically relevant Pum2 target mRNAs. In a collaborative approach with Medenbach (Project 9), we will establish an in vitro translation system from mouse neuronal tissue to recapitulate regulation. The combination with sophisticated mouse models in the Kiebler lab will then allow us to elucidate the underlying mechanisms of Pum2 mediated control of protein synthesis with a special emphasis on local translation at mature synapses and to gain molecular insight into how misregulation results in disease.