Mechanism and targets of Sen1-dependent RNA polymerase II termination

Sen1依赖性RNA聚合酶II终止的机制和靶标

• 批准号: 8044008
• 负责人: DAVID A BROW
• 金额: $ 27.64万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8044008
• 关键词: Active Sites; Alleles; Amino Acids; Amyotrophic Lateral Sclerosis; Ataxia; Bacteria; Base Pairing; Biochemical; Biological Assay; Biological Models; Cellular biology; Code; Collection; Data Set; Defect; Disease; Enzymes; Eukaryota; Foundations; Functional RNA; Fungal Genome; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Suppression; Genetic Transcription; Genome; Goals; Guanosine Triphosphate; Health; Homeostasis; Homologous Gene; Human; Human Biology; Investigation; Lead; Maps; Modeling; Molecular; Molecular Conformation; Muscle Weakness; Mutation; Nerve Degeneration; Neurons; Pathway interactions; Polyadenylation; Polymerase; Process; Protein Analysis; Proteins; Publishing; Quantitative Microscopy; RNA; RNA Polymerase II; RNA-Binding Proteins; Reading; Regulation; Regulator Genes; Saccharomyces cerevisiae; Signal Transduction; Site; Structure; System; Teenagers; Testing; Transcript; Transcription Initiation Site; Transcriptional Regulation; Work; Yeasts; attenuation; base; disabling disease; genetic selection; helicase; human senataxin protein; insight; mathematical model; motor neuron degeneration; mutant; novel; protein function; public health relevance; termination factor; transcription termination

DESCRIPTION (provided by applicant): The realization that non-coding transcripts are remarkably abundant and participate in the regulation of protein- coding genes has revolutionized the study of eukaryotic gene expression. Yet little is known about how non- coding RNAs are made and function. We discovered an RNA polymerase II (Pol II) termination pathway that influences the synthesis and function many non-coding RNAs. This pathway requires the conserved helicase Sen1 and a collection of RNA-binding proteins that recognize the termination signal in the nascent transcript. The Sen1 pathway is essential in the model eukaryote S. cerevisiae (brewer's yeast), and mutations in the human Sen1 gene (SETX) cause the degeneration of motor neurons, leading to forms of ataxia and amyotrophic lateral sclerosis (ALS). Remarkably, Sen1-dependent termination regulates some protein-coding genes by transcription attenuation, which was previously thought to be restricted to bacteria. We will determine the molecular mechanism of Sen1-dependent termination using both genetic and biochemical approaches. Our structure/function studies will focus on two key proteins in the pathway, Sen1 and Pol II, in which we have isolated mutations that result in terminator read-through. Regulation of the IMD2 gene will be examined in detail as a paradigm for NTP homeostasis by Sen1-dependent attenuation and alternative start site selection. We will also identify and characterize new examples of Sen1-dependent gene regulation. The cis- and trans-acting determinants for regulation of these genes will be defined using genetic selections, quantitative gene expression assays, and mathematical modeling. The information gained in these studies will serve as a foundation for the investigation of RNA-based transcriptional regulation in other eukaryotes, including humans. Given the emerging evidence for the importance of non-coding transcripts in gene regulation in humans, our results will have broad implications for eukaryotic biology and human health, and will lead to a better understanding of the causes of motor neuron degeneration. PUBLIC HEALTH RELEVANCE: Mutations in the human Senataxin gene cause motor neuron degeneration that results in crippling muscle weakness, typically starting in the teen years. We discovered that the brewer's yeast homolog of Senataxin, called Sen1, has a crucial function in the regulation of gene expression. We will use yeast as model system to explore Sen1 function and better understand how defects in the human Senataxin protein lead to disabling diseases.

描述（申请人提供）：意识到非编码转录本非常丰富，并且参与蛋白质编码基因的调节已经彻底改变了真核基因表达的研究。然而，关于如何制作非编码RNA和功能知之甚少。我们发现了一个RNA聚合酶II（POL II）终止途径，该途径影响了许多非编码RNA的合成和功能。该途径需要保守的解旋酶SEN1和一组RNA结合蛋白，这些蛋白识别新生转录本中的终止信号。 SEN1途径在酿酒酵母（Brewer's酵母）模型中至关重要，人类SEN1基因（SETX）的突变导致运动神经元的退化，导致共济失调和肌萎缩性侧向硬化症的形式。值得注意的是，SEN1依赖性终止通过转录衰减调节某些蛋白质编码基因，以前被认为仅限于细菌。我们将使用遗传和生化方法来确定SEN1依赖性终止的分子机制。我们的结构/功能研究将集中在途径，SEN1和POL II中的两个关键蛋白上，其中我们具有孤立的突变，从而导致终止读取。 IMD2基因的调节将通过SEN1依赖性衰减和替代起始位点选择来详细研究作为NTP稳态的范例。我们还将确定并表征SEN1依赖性基因调节的新例子。这些基因调节的顺式和反式作用决定因素将使用遗传选择，定量基因表达测定和数学建模来定义。这些研究中获得的信息将成为研究其他真核生物（包括人类）的基于RNA的转录调节的基础。鉴于新兴的证据表明非编码转录本在人类基因调节中的重要性，我们的结果将对真核生物学和人类健康具有广泛的影响，并会更好地理解运动神经元变性的原因。 公共卫生相关性：人类鼻毒素基因的突变会导致运动神经元变性，导致肌肉无力瘫痪，通常从青少年时代开始。我们发现，酿酒剂的塞纳汀（Senataxin）的酵母菌同源物（称为Sen1）在基因表达的调节中具有至关重要的功能。我们将使用酵母作为模型系统来探索SEN1功能，并更好地理解人鼻毒素蛋白中的缺陷如何导致致残疾病。