Exploration of cytoplasmic pre-mRNA splicing in CNS neurons

描述（由申请人提供）：神经系统的发育和功能依赖于其形成特定神经元回路并响应神经元活动和信号传导改变其特性的能力。空间限制性修饰是神经元可塑性调控的关键问题之一。也就是说，需要存在能够局部修饰和生成蛋白质的机制，例如仅在一个突触发生而不在其他突触发生的变化。本研究的目的是探索mRNA的选择性剪接是否发生在神经元细胞质中，并且是否可以通过突触信号传导机制进行局部控制。为了解决这个问题，我们将重点关注神经元细胞表面受体家族（称为神经毒素）的选择性剪接，该受体存在于1，000多种功能不同的剪接变体中。将使用RT-PCR、荧光原位杂交和剪接形式特异性抗体的组合来分析Neurexin剪接。该提案的目的是（1）表征细胞质中含有内含子的neurexin pre-mRNAs，（2）分析可以从神经元细胞质中的pre-mRNAs去除内含子的剪接机制，以及（3）研究neurexin剪接对神经元活性和信号传导的调节。这项研究的发现将提供双重贡献：首先，它们将为neurexin蛋白表达和功能的机制和调控提供新的见解。其次，控制神经元细胞中胞质剪接的主要机制可能不限于neurexin蛋白的剪接，但也可能控制其他神经元蛋白的表达。在突触的局部剪接的特性将支持一种新的机制，调节神经元的可塑性和功能。这些研究的结果与人类健康直接相关。神经节苷脂剪接在缺血中的差异调节。此外，神经毒素的剪接调节神经毒素与两种配体神经配蛋白和α-肌营养不良蛋白聚糖的相互作用。这两种配体都与神经系统疾病有关：精神发育迟滞和自闭症中的神经配素和肌营养不良症中的α-肌营养不良聚糖。因此，在neurexin剪接研究中获得的信息对于了解这些疾病背后的细胞和分子缺陷方面也是有价值的。

DESCRIPTION (provided by applicant): The development and function of the nervous system relies on its ability to form specific neuronal circuits and to alter their properties in response to neuronal activity and signaling. Spatially restricted modification represents one of the key problems in the regulation of neuronal plasticity. That is, mechanisms need to exist that enable the modification and generation of proteins locally, e.g. changes that occur at only one synapse but not at others. The goal of this proposal is to explore whether alternative splicing of mRNAs occurs in the neuronal cytoplasm and can be locally controlled by synaptic signaling mechanisms. To address this question, we will focus on the alternative splicing of a family of neuronal cell surface receptors, called neurexins, which exist in more than 1,000 functionally different splice variants. Neurexin splicing will be analyzed using a combination of RT-PCR, fluorescent in situ hybridization, and splice-form specific antibodies. The aims of this proposal are (1) to characterize cytoplasmic intron-containing neurexin pre-mRNAs, (2) to analyze the splicing machinery that can remove introns from pre-mRNAs in the neuronal cytoplasm, and (3) to investigate the regulation of neurexin splicing in response to neuronal activity and signaling. Findings from this research will provide twofold contributions: First, they will provide novel insights into the mechanisms and regulation of neurexin protein expression and function. Secondly, the principal mechanisms that control cytoplasmic splicing in neuronal cells are likely not restricted to splicing of neurexin proteins but may also control expression of other neuronal proteins. The characterization of local splicing at synapses would support a novel mechanism for regulating neuronal plasticity and function. The findings from these studies have direct relevance for human health. Neurexin splicing is differentially regulated in ischemia. Moreover, splicing of neurexins regulates the interaction of neurexins with two ligands, neuroligins and alpha-dystroglycan. Both ligands have been implicated in nervous system disorders: neuroligins in mental retardation and autism and alpha-dystroglycan in muscular dystrophies. Information obtained in studies on neurexin splicing will therefore also be valuable for understanding aspects of cellular and molecular defects underlying these disorders.