Alternative Splicing of the Drosophilia Dscam Pre-mRNA

描述(申请人提供)：这项建议的长期目标是了解果蝇唐氏综合症细胞黏附分子(DSCAM)基因的选择性剪接是如何调节的，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个外显子是交替剪接的。可选外显子被组织成4个不同的簇，每个簇包含12、48、33和2个互不相容的外显子。因为每个簇内的外显子以相互排斥的方式交替剪接，所以有可能表达38,016种不同的DSCAM亚型。因此，DSCAM是已知的最广泛的选择性剪接基因。Dscam蛋白作为轴突引导受体，在神经发育和功能中发挥重要作用。此外，Dscam还被证明具有免疫受体的功能，帮助机体抵御病原体。目前的证据表明，在单个神经元中表达的亚型的一致性对于神经系统的正确连接至关重要，而在血细胞中表达的异构体对于病原体识别至关重要。因此，了解调控Dscam选择性剪接的机制将有助于深入了解指定果蝇神经元连接和病原体识别的遗传程序。这项建议旨在了解调控DSCAM选择性剪接的机制和DSCAM的互斥剪接机制。首先，我们将确定神经系统外显子4变体在单细胞分辨率下的表达模式，并探索我们在基于组织培养的RNAi筛选中确定的调控蛋白如何控制Dscam在单个神经元中的剪接。第二，我们将解决与互斥剪接机制有关的许多重要问题。具体地说，我们将从功能上剖析RNA二级结构在外显子6相互排斥剪接中的作用，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内含子从前mRNA中移除的顺序，因为这对剪接调控机制有重要影响。我们还将确定一个簇中外显子的剪接是否会影响其他簇中的剪接。最后，我们将比较昆虫和脊椎动物剪接机器处理包含三个或更多相互排斥的外显子簇的前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.