Exploration of cytoplasmic pre-mRNA splicing in CNS neurons

描述(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改变其特性的能力，以响应神经元的活动和信号。空间受限修饰是神经元可塑性调控的关键问题之一。也就是说，需要存在能够在局部修饰和产生蛋白质的机制，例如，只发生在一个突触而不发生在其他突触的变化。这项提议的目的是探索mRNAs的选择性剪接是否发生在神经元细胞质中，并可以受到突触信号机制的局部控制。为了解决这个问题，我们将专注于神经细胞表面受体家族的选择性剪接，称为Neurexins，它存在于1000多种不同功能的剪接变体中。将结合使用RT-PCR、荧光原位杂交和剪接形式特异性抗体来分析Neuresin剪接。本研究的目的是：(1)鉴定胞质内含内含子的neuresin前-mRNAs；(2)分析神经元胞质内含内含子的前-mRNAs的剪接机制；(3)研究neuresin剪接对神经元活动和信号的调节作用。这项研究的发现将提供两方面的贡献：第一，他们将为Neuresin蛋白表达和功能的机制和调控提供新的见解。其次，控制神经细胞胞质剪接的主要机制可能不仅限于Neuresin蛋白的剪接，还可能控制其他神经元蛋白的表达。突触局部剪接的特征将支持一种新的机制来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。神经瑞新剪接在缺血中受到不同的调节。此外，Neurexins的剪接调控Neurexins与两种配体的相互作用，即神经连接蛋白和α-营养不良蛋白聚糖。这两种配体都与神经系统疾病有关：神经连接素在智力低下和自闭症中的作用，以及α-营养不良糖链在肌肉营养不良中的作用。因此，在有关neuresin剪接的研究中获得的信息对于理解这些疾病背后的细胞和分子缺陷方面也将是有价值的。

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.