Alternative Splicing of the Drosophilia Dscam Pre-mRNA

描述（由申请人提供）：本提案的长期目标是了解如何调节果蝇唐氏综合征细胞粘附分子（Dscam）基因的选择性剪接，并确定Dscam选择性剪接相互排斥的机制。Dscam包含115个外显子，其中95个外显子是选择性拼接的。可选外显子被组织成4个不同的集群，每个集群包含12、48、33和2个互斥外显子。由于每个簇内的外显子以互斥的方式选择性拼接，因此有可能表达38,016种不同的Dscam亚型。因此，Dscam是已知的选择性剪接最多的基因。Dscam蛋白作为轴突引导受体在神经发育和功能中起重要作用。此外，Dscam已被证明具有免疫受体的功能，有助于保护生物体免受病原体的侵害。目前的证据表明，在单个神经元中表达的同种异构体的身份对于神经系统的正常连接至关重要，而在血细胞中表达的同种异构体对于病原体识别至关重要。因此，了解调节Dscam选择性剪接的机制将有助于深入了解果蝇神经元连接和病原体识别的遗传程序。本研究旨在了解Dscam的选择性剪接和互斥剪接的调控机制。首先，我们将在单细胞分辨率下确定神经系统中外显子4变异体的表达模式，并探索我们在基于组织培养的RNAi筛选中发现的调节蛋白如何控制果蝇单个神经元中的Dscam剪接。其次，我们将讨论关于互斥剪接机制的许多重要问题。具体来说，我们将从功能上剖析RNA二级结构在外显子6互斥剪接中的作用，并确定其他特征，如距离、剪接位点强度和HRP36结合影响外显子6剪接。此外，我们将确定Dscam内含子从pre-mRNA中去除的顺序，因为这对剪接调节机制具有重要意义。我们还将确定一个簇内外显子的剪接是否会影响其他簇的剪接。最后，我们将比较昆虫和脊椎动物剪接机制处理含有三个或更多互斥外显子簇的前mrna的能力。

DESCRIPTION (provided by applicant): The long term goals of this proposal are to understand how the alternative splicing of the Drosophila Down syndrome cell adhesion molecule (Dscam) gene is regulated and to determine the mechanism by which Dscam alternative splicing is mutually exclusive. Dscam contains 115 exons, 95 of which are alternatively spliced. The alternative exons are organized into 4 distinct clusters containing 12, 48, 33, and 2 mutually exclusive exons each. Because the exons within each cluster are alternatively spliced in a mutually exclusive manner, it is possible that 38,016 different Dscam isoforms can be expressed. Dscam is therefore the most extensively alternatively spliced gene known. The Dscam proteins functions as axon guidance receptors that play an important role in neural development and function. In addition, Dscam has been shown to function as immune receptors that help to defend the organism against pathogens. Current evidence suggests that the identity of the isoforms expressed in individual neurons is critical for proper wiring of the nervous system and in hemocytes is important for pathogen recognition. Thus, understanding the mechanisms regulating Dscam alternative splicing will provide insight into the genetic program that specifies neuronal wiring and pathogen recognition in Drosophila. This proposal is aimed at understanding the mechanisms involved in regulating alternative splicing and the mechanism of mutually exclusive splicing of Dscam. First, we will determine the expression pattern of the exon 4 variants in the nervous system at single cell resolution and explore how regulatory proteins we have identified in tissue culture-based RNAi screens control Dscam splicing in individual neurons in the fly. Second, we will address many important issues regarding the mechanisms of mutually exclusive splicing. Specifically, we will functionally dissect the role of RNA secondary structures in exon 6 mutually exclusive splicing and identify other features such as distance, splice site strength, and HRP36 binding impact exon 6 splicing. Moreover, we will determine the order in which the Dscam introns are removed from the pre-mRNA as this has important implications on the splicing regulatory mechanisms. We will also determine if the splicing of exons within one cluster impacts splicing in other clusters. Finally, we will compare the ability of the insect and vertebrate splicing machinery to process pre-mRNAs containing clusters of three or more mutually exclusive exons. PUBLIC HEALTH RELEVANCE: These experiments will provide tremendous insight into the mechanisms of alternative splicing and mutually exclusive splicing. As the vast majority of human genes are alternatively spliced, it is likely the discoveries we make will be of direct relevance to human health. Moreover, the results will provide significant insight into the mechanisms of neural wiring and immunity.