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果蝇唐氏综合征细胞粘附分子（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个是选择性剪接的。替代外显子被组织成4个不同的簇，每个簇包含12、48、33和2个相互排斥的外显子。由于每个簇内的外显子以互斥的方式选择性剪接，因此可以表达38，016种不同的Dscam同种型。因此，Dscam是已知的最广泛的选择性剪接基因。Dscam蛋白作为轴突导向受体发挥功能，在神经发育和功能中发挥重要作用。此外，Dscam已被证明可以作为免疫受体，帮助保护生物体免受病原体的侵害。目前的证据表明，个体神经元中表达的同种型的身份对于神经系统的正确布线至关重要，并且在血细胞中对于病原体识别很重要。因此，了解Dscam选择性剪接的调控机制，将有助于深入了解果蝇中指定神经元布线和病原体识别的遗传程序。该建议旨在了解参与调节选择性剪接的机制和Dscam互斥剪接的机制。首先，我们将在单细胞分辨率下确定神经系统中外显子4变体的表达模式，并探索我们在基于组织培养的RNAi筛选中鉴定的调控蛋白如何控制果蝇个体神经元中的Dscam剪接。第二，我们将解决许多关于互斥拼接机制的重要问题。具体来说，我们将功能性地剖析RNA二级结构在外显子6互斥剪接中的作用，并确定其他功能，如距离，剪接位点强度和HRP 36结合影响外显子6剪接。此外，我们将确定Dscam内含子从前体mRNA中去除的顺序，因为这对剪接调控机制具有重要意义。我们还将确定一个簇内外显子的剪接是否影响其他簇中的剪接。最后，我们将比较昆虫和脊椎动物的剪接机制的能力，以处理前mRNA包含集群的三个或更多个互斥的外显子。 公共卫生相关性：这些实验将提供巨大的洞察机制的选择性剪接和互斥剪接。由于绝大多数人类基因是选择性剪接的，我们的发现很可能与人类健康直接相关。此外，这些结果将为神经布线和免疫机制提供重要的见解。

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.