Hu proteins as novel splicing regulators in neurons

描述(申请人提供)：该项目的长期目标是研究HU家族副肿瘤性神经疾病(PND)抗原在神经元中作为选择性剪接调节因子的一种新功能。HU蛋白是用PND患者血清克隆的一组神经元RNA结合蛋白，是由小细胞肺癌引发的HU综合征的抗原。与它们最接近的果蝇同源物ELAV相似，HU蛋白被证明是哺乳动物神经系统中神经元分化所必需的。然而，HU蛋白作为RNA结合蛋白的功能尚不清楚。除了少数例子表明它们与3‘-未翻译区的富含AU元件的mRNA结合来调节mRNA的稳定性和翻译外，HU蛋白在体内的靶点很大程度上是未知的。最近，我们已经确定了HU蛋白的两个RNA靶标，并为这些蛋白质作为替代剪接调节因子的突出作用提供了令人信服的证据。为了表征Hu蛋白在神经元中作为剪接调节因子的新功能，我们提出了三个特定的目标。在目标I和II中，我们将研究控制HU蛋白对两个新发现的含有HU靶标的前mRNAs的神经元特异性选择性RNA加工的机制。关于HU蛋白功能的具体假设将被产生，并通过体外和体内实验进行测试。在AIM III中，我们将使用电子数据库筛选和基因组SELEX方法来识别更多与HU蛋白结合的神经元靶点。确定这些新的目标将为今后的工作奠定基础，这些工作将侧重于拟议研究中提出的原则的普遍性。这些研究不仅将对HU蛋白在神经元发育和功能中的作用提供重要的见解，而且将为PND疾病--HU综合征的发病机制提供重要的线索。最后，这些研究还将极大地增加我们对神经元选择性剪接控制机制的理解。

DESCRIPTION (provided by applicant): The long-term goal of this project is to investigate a novel function of the Hu family of paraneoplastic neurological disease (PND) antigens in neurons as alternative splicing regulators. Hu proteins belong to a group of neuronal RNA-binding proteins that were cloned using antiserum from PND patients and are antigens of the Hu syndrome triggered by small cell lung carcinomas. Similar to their closest Drosophila homolog ELAV, Hu proteins were shown to be required for neuronal differentiation in the mammalian nervous system. However, the function of Hu proteins as RNA-binding proteins is not understood. Except for a handful of examples indicating that they bind to the AU-rich element of 3'-untanslated region of mRNA to regulate mRNA stability and translation, the in vivo targets of Hu proteins are largely unknown. Recently we have identified two RNA targets for Hu proteins and provided compelling evidence for a prominent role of these proteins as alternative splicing regulators. To characterize the novel function of Hu proteins as splicing regulators in neurons, we propose three specific aims. In aims I and II, we will study the mechanisms that control the neuron-specific alternative RNA processing of the two newly identified Hu target-containing pre-mRNAs by Hu proteins. Specific hypotheses regarding the function of Hu proteins will be generated and tested with both in vitro and in vivo experiments. In aim III, we will identify additional neuronal targets bound by Hu proteins using both in silico database screening and genomic SELEX approaches. The identification of these novel targets will provide the basis for future work that will focus on universality of the principle developed in the proposed studies. These studies will provide not only significant insights into the role of Hu proteins in the development and function of neurons, but also important hints of pathogenesis of the PND disease, the Hu syndrome. Finally, these studies will also greatly increase our understanding of the mechanisms controlling alternative splicing in neurons.