Hu proteins as novel splicing regulators in neurons

描述（由申请人提供）：本项目的长期目标是研究副肿瘤性神经疾病（PND）抗原的Hu家族作为选择性剪接调节剂在神经元中的新功能。Hu蛋白属于使用来自PND患者的抗血清克隆的一组神经元RNA结合蛋白，并且是由小细胞肺癌触发的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。除了少数实例表明它们与mRNA的3 '-非翻译区的富含AU的元件结合以调节mRNA的稳定性和翻译外，Hu蛋白的体内靶点在很大程度上是未知的。最近，我们已经确定了两个RNA目标的胡蛋白，并提供了令人信服的证据，这些蛋白质作为选择性剪接调节剂的突出作用。为了表征Hu蛋白作为神经元中剪接调节剂的新功能，我们提出了三个具体目标。在目标I和II中，我们将研究Hu蛋白控制两个新鉴定的含Hu靶的前mRNA的神经元特异性替代RNA加工的机制。将产生关于Hu蛋白功能的具体假设，并通过体外和体内实验进行测试。在目标III中，我们将使用计算机数据库筛选和基因组SELEX方法鉴定Hu蛋白结合的其他神经元靶点。这些新目标的确定将为未来的工作提供基础，这些工作将侧重于拟议研究中开发的原理的普遍性。这些研究不仅将为Hu蛋白在神经元发育和功能中的作用提供重要的见解，而且还将为PND疾病Hu综合征的发病机制提供重要的线索。最后，这些研究也将大大增加我们对神经元中控制选择性剪接的机制的理解。

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.