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的能力，并根据神经元活动和信号改变其特性。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，只发生在一个突触上而不发生在其他突触上的变化。本研究的目的是探讨mrna的选择性剪接是否发生在神经元细胞质中，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的选择性剪接，称为神经素，它存在于1000多种功能不同的剪接变体中。Neurexin剪接将使用RT-PCR，荧光原位杂交和剪接形式特异性抗体的组合进行分析。本研究的目的是：(1)表征胞质中含有内含子的神经rexin pre- mrna，(2)分析可以从神经元细胞质中的pre- mrna中去除内含子的剪接机制，以及(3)研究神经rexin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两方面的贡献：首先，它们将为神经rexin蛋白表达和功能的机制和调控提供新的见解。其次，控制神经元细胞细胞质剪接的主要机制可能不仅限于神经rexin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调节神经元可塑性和功能的新机制。这些研究的结果与人类健康直接相关。缺血时，Neurexin剪接受到差异调节。此外，神经素的剪接调节了神经素与两种配体的相互作用，即神经素和α -歧化聚糖。这两种配体都与神经系统疾病有关：神经素与智力迟钝和自闭症有关，α -糖营养不良与肌肉营养不良有关。因此，在神经蛋白剪接研究中获得的信息也将对理解这些疾病背后的细胞和分子缺陷方面有价值。

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.