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

摘要 肌醇焦磷酸（inositolpyrophosphates，IPPs）是从端粒开始参与多种细胞过程的信号分子 维持和凋亡到囊泡运输和细胞迁移。IPP水平的改变（通过 IPP代谢酶）与人类病理学有关，包括癌症、肥胖症、糖尿病和听力损失。 多效性效应表明肌醇焦磷酸有能力控制非常基本的细胞凋亡， 功能协调发展的IPPs在磷酸盐传感和磷酸盐稳态中的作用在真菌、植物和植物中有记载。 人类细胞通过诱导磷酸盐获得基因的转录来响应磷酸盐限制，然而， 在每个分类群中实现这一点的机制是不同的。 粟酒裂殖酵母包含三个基因，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。pho 1的表达， pho 84和tgp 1在富含磷酸盐的培养基中生长期间被RNA聚合酶II（Pol 2）主动抑制 顺式转录来自相应5'侧翼基因prt、prt 2和nc-tgp 1的长非编码（lnc）RNA。 通过PHO mRNA启动子的lncRNA转录取代了激活转录因子Pho 7， 从而干扰PHO mRNA表达。lncRNA介导的转录干扰系统是 对影响3 '-加工/终止的遗传操作敏感。引发“早熟”的突变 lncRNA 3 '-加工/终止响应于mRNA启动子上游的poly（A）信号，导致去 抑制pho 1，而减少lncRNA终止的遗传变化过度抑制pho 1表达。 我们已经利用这个系统来发现对3 '-加工和Pol 2终止的新影响。我们展示 （i）lncRNA转录受肌醇焦磷酸1，5-IP 8的代谢物控制， 早熟3 '-加工/终止的激动剂;（ii）Spx 1，其由SPX结构域和RING E3组成 连接酶结构域，是IP 8信号传导至3 '-加工/终止机制的可能介质;（iii）14-3-3 蛋白Rad 24以依赖于其磷酸盐的方式拮抗早熟的3 '-加工/终止 结合位点;和（iv）Pol 2终止可以通过在必需的蛋白质中的功能获得性突变来增强。 终止因子Seb 1。具体目的是：（1）鉴定Spx 1 E3泛素连接酶的靶点（通过TULIP 2 标记和质谱法）;并测试是否焦磷酸化的组分（或IP 8结合）， Pol 2机制有助于IP 8对3 '-加工/终止的影响;（2）利用新的分析方法， 评估遗传学预测的裂殖酵母菌株中IPP丰度和同种型分布的方法 IPP含量的变化，并研究磷酸盐饥饿是否以及如何影响细胞IPP含量; (3)为了探索Rad 24拮抗3 '-加工/终止的机制，通过进行ChIP-seq， 鉴定与Rad 24在体内相关的基因;和（4）在细胞中产生新的功能获得性突变， 分析了它们对poly（A）位点利用、RNA结合、CTD结合的影响。

ABSTRACT 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. A role for IPPs in phosphate sensing and phosphate homeostasis is documented in fungi, plants, and humans. Cells respond to phosphate limitation by inducing the transcription of phosphate acquisition genes, yet the mechanisms by which this is achieved differ in each taxon. The phosphate (PHO) 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 RNA polymerase II (Pol2) transcription in cis of a long noncoding (lnc) RNA from the respective 5' flanking genes prt, prt2, and nc-tgp1. lncRNA transcription across the PHO mRNA promoter displaces the activating transcription factor Pho7 and thereby interferes with PHO mRNA expression. The system of lncRNA-mediated transcriptional interference is sensitive to genetic manipulations that influence 3’-processing/termination. Mutations that elicit “precocious” lncRNA 3'-processing/termination in response to poly(A) signals upstream of the mRNA promoters lead to de- repression of pho1, whereas genetic changes that diminish lncRNA termination hyper-repress pho1 expression. We have exploited this system to discover novel influences on 3’-processing and Pol2 termination. We showed that: (i) lncRNA transcription is subject to metabolite control by inositol pyrophosphate 1,5-IP8, which acts as an agonist of precocious 3'-processing/termination; (ii) Spx1, which is composed of an SPX domain and a RING E3 ligase domain, is a likely mediator of IP8 signaling to the 3'-processing/termination machinery; (iii) the 14-3-3 protein Rad24 antagonizes precocious 3’-processing/termination in a manner that depends on its phosphate binding site; and (iv) Pol2 termination can be enhanced via a gain-of-function mutation in the essential termination factor Seb1. Specific aims are: (1) to identify targets of the Spx1 E3 ubiquitin ligase (via TULIP2 tagging and mass spectrometry); and to test whether pyrophosphorylation of (or IP8 binding to) components of the Pol2 machinery contributes to the effects of IP8 on 3’-processing/termination; (2) to leverage new analytical methods to assess IPP abundance and isoform distribution in fission yeast strains where genetics predicts changes in IPP content, and to investigate whether and how phosphate starvation impacts cellular IPP content; (3) To probe the mechanism by which Rad24 antagonizes 3’-processing/termination, by performing ChIP-seq to identify genes with which Rad24 is associated in vivo; and (4) To generate new gain-of-function mutations in the essential termination factor Seb1 and analyze their effects on poly(A) site usage, RNA binding, CTD binding.