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

基本信息

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
项目类别：
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&apos Splice Site 5&apos 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

项目摘要

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： “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：真核基因表达受真核生物之间功能耦合的复杂网络的调节。哺乳动物原钙粘蛋白 (Pcdh) 的转录起始、延伸和 RNA 加工过程。基因簇提供了一个显着且极其重要的系统来研究潜在的 Pcdh 基因簇编码的蛋白质在这一耦合过程的机制中发挥着重要作用。在神经回路组装中，通过为单个神经元提供独特的细胞表面“代码”来形成 Pcdh 细胞表面代码是通过复杂的随机过程生成的。启动子选择、选择性 RNA 剪接以及细胞中 Pcdh 顺式二聚体的组合组装转录过程涉及单个 Pcdh 基因启动子的“随机”激活。通过长程增强子-启动子 DNA 环化（需要 DNA 结合蛋白 CTCF），转录多达 250,000 个 DNA 碱基对，然后剪接启动子近端 5' 剪接尽管在理解基因组方面已经取得了重大进展。簇状 Pcdh 的 DNA 组织、单个神经元表达和染色体结构域配置，转录起始和延伸以及RNA加工耦合的机制仍然存在最近的研究揭示了高度保守的 RNA 双链体的显着组织。 Pcdh pre-mRNA 中的结构，以及 Pcdh 启动子处的聚合转录的惊人模式。这些初步观察结果得出了一个模型，其中这些 RNA 二级结构调节 5' 该提案的目标 1 是确定剪接位点选择，以及启动子选择的新机制。哺乳动物细胞中 Pcdh α 和 γ RNA 前体的结构并研究它们的功能，目标 2 目的是确定 CTCF 在调节 Pcdhα RNA 的多种转录和加工中的作用。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探针和 RNASeq 方法、单分子可视化方法进行结构成像 RNA聚合酶在基因簇中进行时的易位，以及基因编辑方法确定转录和剪接所需的调控元件。拟议的研究准备进行。揭示了真核基因调控的新颖且令人兴奋的调控机制。蛋白质在神经回路组装中发挥核心作用，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可以提供基本的见解基因表达的新机制，也使人们更好地了解基因的遗传基础因此，拟议的研究与人类健康直接相关。