Mechanism and targets of Sen1-dependent RNA polymerase II termination

描述（由申请人提供）：认识到非编码转录物非常丰富并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，人们对非编码RNA是如何产生和发挥作用的却知之甚少.我们发现了RNA聚合酶II（Pol II）终止途径，该途径影响许多非编码RNA的合成和功能。该途径需要保守的解旋酶Sen 1和一系列RNA结合蛋白，这些蛋白识别新生转录物中的终止信号。Sen 1通路在模式真核生物S.酿酒酵母（啤酒酵母）中的突变和人Sen 1基因（SETX）中的突变引起运动神经元的变性，导致共济失调和肌萎缩性侧索硬化（ALS）的形式。值得注意的是，Sen 1依赖性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。我们将使用遗传和生物化学方法来确定Sen 1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中的两个关键蛋白质，Sen 1和Pol II，其中我们已经分离出导致终止子通读的突变。IMD 2基因的调节将作为NTP稳态的范例通过Sen 1依赖性衰减和替代起始位点选择进行详细检查。我们还将确定和表征Sen 1依赖性基因调控的新实例。将使用遗传选择、定量基因表达测定和数学建模来定义用于调节这些基因的顺式和反式作用决定簇。在这些研究中获得的信息将作为在其他真核生物，包括人类的RNA为基础的转录调控的调查的基础。鉴于非编码转录本在人类基因调控中的重要性的新证据，我们的研究结果将对真核生物学和人类健康产生广泛的影响，并将导致更好地了解运动神经元变性的原因。 公共卫生相关性：人类Senataxin基因的突变会导致运动神经元变性，导致严重的肌肉无力，通常始于青少年时期。我们发现，啤酒酵母Senataxin同系物，称为Sen 1，在基因表达的调节中具有关键作用。我们将使用酵母作为模型系统来探索Sen 1功能，并更好地了解人类Senataxin蛋白的缺陷如何导致致残性疾病。

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.