Coupling promoter choice and alternative RNA splicing in the mammalian Protocadherin gene cluster

标题： “哺乳动物原钙粘蛋白基因簇中的偶联启动子选择和选择性RNA剪接” 项目概要： 真核基因的表达是由一个复杂的网络的功能耦合之间的调控， 转录起始、延伸和RNA加工的过程。原钙粘蛋白（Protocadherin，Pcdh） 基因簇提供了一个显着的和根本重要的系统来研究潜在的 这一耦合过程的机制。由Pcdh基因簇编码的蛋白质起着重要作用 在神经电路组装中，通过为单个神经元提供独特的细胞表面“代码”， 自我认知的基础。Pcdh细胞表面代码是由一个复杂的随机过程产生的。 启动子选择、选择性RNA剪接和Pcdh顺式二聚体在细胞中的组合组装 面转录过程涉及单个Pcdh基因启动子的“随机”激活 通过长距离增强子-启动子DNA环（其需要DNA结合蛋白CTCF）， 转录多达250，000个碱基对的DNA，随后剪接启动子近端5'剪接 位点连接到远处的3'剪接位点。尽管在理解基因组方面已经取得了重大进展， 聚簇Pcdh的DNA组织、单个神经元表达和染色体结构域构型， 转录起始和延伸以及RNA加工的耦合机制仍然存在 未知最近的研究揭示了高度保守的RNA双链体的显着组织 Pcdh前体mRNA中的结构，以及Pcdh启动子处的会聚转录的显著模式。 这些初步的观察结果导致了一个模型，其中这些RNA二级结构调节5'端的转录。 剪接位点选择以及启动子选择的新机制。本提案的目标1是确定 Pcdh α和γ RNA前体在哺乳动物细胞中的结构并研究其功能，目的2 目的：研究CTCF在Pcdhα RNA转录和加工中的作用。各种 方法将被用来实现这些目标，包括在体内分析的RNA二级 使用化学探针和RNASeq方法，单分子可视化方法来成像 RNA聚合酶在通过基因簇时的易位，以及基因编辑方法， 鉴定转录和剪接所需的调控元件。拟议中的研究准备 揭示了新的和令人兴奋的真核基因调控机制。此外，作为Pcdh 蛋白质在神经回路组装中起着核心作用，并且它们与神经系统疾病有关。 疾病，了解Pcdh基因表达的细节不仅将提供基本的见解， 新的基因表达机制，而且还导致更好地了解基因表达的遗传基础。 神经系统疾病，如自闭症。因此，拟议的研究与人类健康直接相关。

TITLE: “Coupling promoter choice and alternative RNA splicing in the mammalian Protocadherin gene cluster” PROJECT SUMMARY: Eukaryotic gene expression is regulated by a complex network of functional coupling between the processes of transcription initiation, elongation and RNA processing. The mammalian Protocadherin (Pcdh) gene cluster provides a remarkable and fundamentally important system to study the underlying mechanisms of this coupling process. The proteins encoded by the Pcdh gene cluster play an essential role in neural circuit assembly by providing individual neurons with a unique cell surface “code” that forms the basis of self-recognition. The Pcdh cell surface code is generated by a complex process of stochastic promoter choice, alternative RNA splicing and combinatorial assembly of Pcdh cis-dimers at the cell surface. The transcriptional process involves “stochastic” activation of individual Pcdh gene promoters through long-range enhancer-promoter DNA looping (which requires the DNA binding protein CTCF), transcription of as much as 250,000 base pairs of DNA, followed by splicing of a promoter proximal 5' splice site to a distant 3' splice site. Although significant progress has been made in understanding the genomic DNA organization, single neuron expression, and chromosome domain configuration of the clustered Pcdh, the mechanisms by which transcriptional initiation and elongation, and RNA processing are coupled remain unknown. Recent studies have revealed a remarkable organization of highly conserved RNA duplex structures in the Pcdh pre-mRNAs, and a striking pattern of convergent transcription at Pcdh promotors. These preliminary observations have lead to a model in which these RNA secondary structures regulate 5' splice site choice, and a novel mechanism for promoter choice. Aim 1 of this proposal is to Determine the architecture of Pcdh α and γ RNA precursors in mammalian cells and investigate their functions, and Aim 2 is to Determine the role of CTCF in regulating transcription and processing of Pcdhα RNAs. A variety of approaches will be used to accomplish these aims, including the in vivo analysis of RNA secondary structures using chemical probes and RNASeq methods, single molecule visualization methods to image the translocation of RNA polymerase as it proceeds through the gene cluster, and gene editing methods to identify regulatory elements required for transcription and splicing. The proposed studies are poised to reveal novel and exciting regulatory mechanisms governing eukaryotic gene regulation. Moreover, as Pcdh proteins play a central role in neural circuit assembly, and they have been implicated in neurological diseases, understanding the details of Pcdh gene expression will not only provide fundamental insights into novel mechanisms of gene expression, but also lead to a better understanding of the genetic basis of neurological diseases, such as autism. Thus, the proposed research is of direct relevance to human health.