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

项目摘要 肌醇焦磷酸是参与多种细胞过程的信号分子 从端粒维持和凋亡到囊泡运输和细胞迁移。改变 IPP水平（通过IPP代谢酶的突变）与人类病理学有关，包括 癌症、肥胖症、糖尿病和听力损失。这种多效性表明肌醇 焦磷酸盐具有控制非常基本的细胞功能的能力。众所周知，IPP 在酵母、植物和哺乳动物中参与磷酸盐感应和磷酸盐稳态 细胞真菌通过诱导磷酸盐的转录来响应磷酸盐饥饿 获得基因裂殖酵母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Pho 1、 无机磷酸盐转运蛋白Pho 84和甘油磷酸盐转运蛋白Tgp 1。表达 pho 1，pho 84和tgp 1在富磷培养基中的生长过程中被 顺式转录来自相应5 ′侧翼基因prt，prt 2， 和NC-TGP 1。这表明上游lncRNA的转录干扰表达 下游的mRNA基因的取代激活转录因子Pho 7从其 mRNA启动子中的结合位点。本提案的主要发现是 我们的发现是：（i）3 '-加工和转录终止是lncRNA中的控制点， 介导的3 ′-侧翼基因表达的抑制，和（ii）来自prt-pho 1的Pho 1表达 基因座是细胞对终止的影响的敏感读数。基于这些发现，我们 假设IPP动力学影响3 ′-加工/转录终止和影响 聚（A）位点使用。具体目标是：（1）使用遗传阵列分析，并揭示程度， 其中3'加工/转录终止因子，RNA Pol II CTD，和 参与塑造CTD磷酸化阵列的因素取决于IPP水平;（2）评估- 在全基因组水平-IPP动力学对基因表达和3 '端形成的影响， 通过分析mRNA和新生RNA谱，并绘制野生型细胞和非野生型细胞中的poly（A）位点， IPP水平改变的细胞;（3）探索IPP影响Pol 2的机制 转录终止使用体外合成的IPP，我们将测试是否组分的 3 '-加工/转录终止机制是焦磷酸化的靶点， IPP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 参与裂殖酵母磷酸盐稳态信号转导途径。

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.