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

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

• 批准号: 9904728
• 负责人: Daniele Canzio
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9904728
• 关键词: 3' Splice Site; 5' Splice Site; Adenosine; Alternative Splicing; Antisense RNA; Architecture; Base Pairing; Binding; Binding Sites; Biochemical; Biology; Brain; CCCTC-binding factor; Cell surface; Chemicals; Chromatin; Chromatin Loop; Chromosome Structures; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Complex; Coupled; Couples; Coupling; Cytidine; DNA; DNA-Directed RNA Polymerase; Data; Development; Distant; Enhancers; Ensure; Exons; Gene Activation; Gene Cluster; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Transcription; Genomic DNA; Genomics; Goals; Grant; Health; High-Throughput Nucleotide Sequencing; Human; Human Genome; Image; Individual; K-Series Research Career Programs; Laboratories; Lead; Mammalian Cell; Mammals; Measures; Mediating; Mentors; Methods; Modeling; Modification; Molecular; Molecular Conformation; Neurons; Nucleotides; Pattern; Phase; Play; Polychlorinated Biphenyls; Process; Protein Isoforms; Proteins; RNA; RNA Precursors; RNA Processing; RNA Splicing; RNA analysis; Regulation; Regulatory Element; Research; Role; Spliceosomes; Stochastic Processes; Structure; System; Testing; Thermodynamics; Training; Transcript; Transcription Elongation; Transcription Initiation; Transcription Initiation Site; Transcription Process; Visualization; autism spectrum disorder; combinatorial; dimer; dimethyl sulfate; experimental study; fascinate; in silico; in vivo; insight; mRNA Precursor; nervous system disorder; neural circuit; neuronal circuitry; novel; prevent; promoter; single molecule; transcriptome sequencing

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.

标题： “耦合启动子的选择和哺乳动物方案her蛋白基因簇中的替代RNA剪接” 项目摘要： 真核基因表达受到复杂的功能耦合网络的调节 转录启动，伸长和RNA处理的过程。哺乳动物协议（PCDH） Gene群集提供了一个非凡且根本重要的系统来研究基础 这个耦合过程的机制。 PCDH基因簇编码的蛋白质起着至关重要的作用 在神经元组件中，通过为单个神经元提供独特的细胞表面“代码”，形成了 自我认识的基础。 PCDH细胞表面代码是由随机的复杂过程生成的 启动子选择，替代RNA剪接和PCDH顺式二聚体组合的组合组合 表面。转录过程涉及单个PCDH基因启动子的“随机”激活 通过远程增强子启动器DNA循环（需要DNA结合蛋白CTCF）， 转录多达250,000个碱基对DNA，然后剪接启动子代理5'剪接 位于遥远的3'剪接站点。尽管在理解基因组方面取得了重大进展 DNA组织，单神经元表达和聚类PCDH的染色体结构域配置， 连接转录启动和伸长和RNA处理的机制仍然存在 未知。最近的研究表明，高度保守的RNA双链体组织 PCDH前MRNA中的结构，以及PCDH启动子的趋同转录模式。 这些初步观察结果导致了这些RNA二次结构调节5'的模型 剪接网站选择，以及一种新型的启动子选择机制。该提议的目标1是确定 PCDHα和γRNA前体在哺乳动物细胞中的结构并研究其功能，并瞄准2 是确定CTCF在PCDHαRNA的调节转录和处理中的作用。各种各样 方法将用于实现这些目标，包括RNA次级的体内分析 使用化学问题和RNASEQ方法的结构，单分子可视化方法图像 RNA聚合酶通过基因簇进行的易位，以及基因编辑方法 确定转录和剪接所需的调节元素。拟议的研究中毒 揭示有关真核基因调节的新颖而令人兴奋的调节机制。而且，作为pcdh 蛋白质在神经元电路组件中起着核心作用，并且在神经学中隐含 疾病，了解PCDH基因表达的细节不仅将提供基本的见解 基因表达的新型机制，但也可以更好地理解 神经疾病，例如自闭症。这是拟议的研究与人类健康的直接相关。