Inositol pyrophosphate dynamics affect RNA 3'-processing/transcription termination

项目总结 焦磷酸肌醇(Ipps)是参与多种细胞过程的信号分子。 从端粒维持和细胞凋亡到囊泡运输和细胞迁移。改建 IPP水平(通过IPP代谢酶的突变)与人类病理有关，包括 癌症、肥胖症、糖尿病和听力损失。多效性表明肌醇 焦磷酸盐有能力控制非常基本的细胞功能。IPP是众所周知的 参与酵母、植物和哺乳动物的磷酸盐传感和磷酸盐稳态 细胞。真菌对磷饥饿的反应是通过诱导磷的转录来实现的 获取基因。裂殖酵母庞贝裂殖酵母中的磷酸调节子 包括三个基因，分别指定细胞表面酸性磷酸酶Pho1，和 无机磷转运蛋白Pho84和甘油磷酸转运蛋白Tgp1。的表达 在富磷介质中的生长过程中，Pho1、Pho84和tgp1被主动抑制。 分别来自5‘侧翼基因prt，prt2， 和nc-tgp1。有人认为，上游lncrna的转录干扰了表达。 通过取代激活转录因子Pho7来改变下游的mRNA基因 M RNA启动子中的结合位点(S)。本提案背后的主要发现是 我们的发现是：(I)3‘-加工和转录终止是lncRNA- 介导的3‘-侧翼基因表达的抑制，以及(Ii)PrT-Pho1的Pho1表达 基因座是细胞影响终止的灵敏读数。基于这些发现，我们 假设IPP动力学影响3‘-加工/转录终止及其影响 Poly(A)站点使用量。具体目标是：(1)使用基因阵列分析并揭示 其中3‘加工/转录终止因子、RNA POL II CTD和 塑造CTD磷酸化阵列所涉及的因素取决于IPP水平；(2)评估- 在全基因组水平上--IPP动态对基因表达和3‘末端形成的影响， 通过分析信使核糖核酸和新生核糖核酸图谱并定位野生型细胞和 IPP水平改变的细胞；以及(3)探索IPP影响Pol2的机制 转录终止。使用体外合成的IPP，我们将测试 3‘-加工/转录终止机制是焦磷酸化和 IPPs是否影响CTD激酶的活性。我们期望获得新的和普遍的见解 这些重要的信号分子在基因表达中的作用，并阐明 信号转导通路参与裂解酵母磷稳态。

Project summary Inositol pyrophosphates (IPPs) are signaling molecules involved in diverse cellular processes from telomere maintenance and apoptosis to vesicular trafficking and cell migration. Alterations in IPP levels (via mutations in IPP metabolizing enzymes) are linked to human pathology including cancer, obesity, diabetes and hearing loss. The pleiotropic effects suggest that inositol pyrophosphates have the ability to control very basic cellular functions. IPPs are known to participate in phosphate sensing and phosphate homeostasis in yeast, plant and mammalian cells. Fungi respond to phosphate starvation by inducing the transcription of phosphate acquisition genes. The phosphate regulon in the fission yeast Schizosaccharomyces pombe comprises three genes that specify, respectively, a cell surface acid phosphatase Pho1, an inorganic phosphate transporter Pho84, and a glycerophosphate transporter Tgp1. Expression of pho1, pho84, and tgp1 is actively repressed during growth in phosphate-rich medium by the transcription in cis of a long noncoding (lnc) RNA from the respective 5' flanking genes prt, prt2, and nc-tgp1. It is proposed that transcription of the upstream lncRNA interferes with expression of the downstream mRNA genes by displacing the activating transcription factor Pho7 from its binding site(s) in the mRNA promoters. The key discoveries underlying the present proposal are our findings that: (i) 3’-processing and transcription termination is a control point in the lncRNA- mediated repression of 3’-flanking gene expression, and (ii) Pho1 expression from the prt–pho1 locus is a sensitive read-out of cellular influences on termination. Based on these findings, we hypothesize that IPP dynamics affect 3’-processing/transcription termination and influence poly(A) site usage. Specific aims are to: (1) use genetic array analyses and reveal the extent to which the functions of 3’ processing/transcription termination factors, the RNA Pol II CTD, and factors involved in sculpting the CTD phosphorylation array depend on IPP levels; (2) assess – at the genome-wide level – the impact of IPP dynamics on gene expression and 3’-end formation, by analyzing mRNA and nascent RNA profiles and mapping poly(A) sites in wild-type cells and in cells with altered IPP levels; and (3) explore mechanisms by which IPPs influence Pol2 transcription termination. Using in vitro synthesized IPPs, we will test whether components of the 3’-processing/transcription termination machinery are targets for pyrophosphorylation and whether IPPs affect the activities of CTD kinases. We expect to gain new and general insights into the role of these important signaling molecules in gene expression, and to illuminate the signal transduction pathway involved in fission yeast phosphate homeostasis.