Mechanism and targets of Sen1-dependent RNA polymerase II termination

描述(申请人提供)：认识到非编码转录本非常丰富，并参与蛋白质编码基因的调控，使真核基因表达的研究发生了革命性的变化。然而，关于非编码RNA是如何制造和发挥作用的，人们知之甚少。我们发现了一个影响许多非编码RNA合成和功能的RNA聚合酶II(POL II)终止途径。这一途径需要保守的解旋酶Sen1和一系列识别新生转录本中终止信号的RNA结合蛋白。Sen1通路在真核生物酿酒酵母模型中是必不可少的，人类Sen1基因(SETX)的突变会导致运动神经元的退化，导致共济失调和肌萎缩侧索硬化症(ALS)的形成。值得注意的是，Sen1依赖的终止通过转录衰减来调节一些蛋白质编码基因，这在以前被认为仅限于细菌。我们将用遗传学和生物化学方法来确定依赖Sen1终止的分子机制。我们的结构/功能研究将集中在该途径中的两个关键蛋白质Sen1和POL II上，在这两个蛋白质中，我们分离出了导致终结者通读的突变。IMD2基因的调节将作为NTP动态平衡的范例，通过依赖于Sen1的衰减和替代的起点选择进行详细的研究。我们还将识别和表征依赖于Sen1的基因调控的新例子。调控这些基因的顺式和反式决定因素将使用遗传选择、定量基因表达分析和数学建模来定义。从这些研究中获得的信息将作为研究其他真核生物，包括人类的基于RNA的转录调控的基础。鉴于越来越多的证据表明非编码转录本在人类基因调控中的重要性，我们的结果将对真核生物学和人类健康产生广泛的影响，并将有助于更好地理解运动神经元变性的原因。 与公共健康相关：人类感觉神经毒素基因的突变会导致运动神经元退化，导致严重的肌肉无力，通常从十几岁开始。我们发现啤酒酿造商的酵母同系物Senataxin，称为Sen1，在基因表达调控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的功能，并更好地了解人类Senataxin蛋白的缺陷是如何导致致残性疾病的。

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.