Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA.
Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.
Nucleic Acids Res. 2020 Nov 4;48(19):10739-10752. doi: 10.1093/nar/gkaa776.
Fission yeast phosphate homeostasis genes are repressed in phosphate-rich medium by transcription of upstream lncRNAs that interferes with activation of the flanking mRNA promoters. lncRNA control of PHO gene expression is influenced by the Thr4 phospho-site in the RNA polymerase II CTD and the 3' processing/termination factors CPF and Rhn1, mutations of which result in hyper-repression of the PHO regulon. Here, we performed a forward genetic screen for mutations that de-repress Pho1 acid phosphatase expression in CTD-T4A cells. Sequencing of 18 independent STF (Suppressor of Threonine Four) isolates revealed, in every case, a mutation in the C-terminal pyrophosphatase domain of Asp1, a bifunctional inositol pyrophosphate (IPP) kinase/pyrophosphatase that interconverts 5-IP7 and 1,5-IP8. Focused characterization of two STF strains identified 51 coding genes coordinately upregulated vis-à-vis the parental T4A strain, including all three PHO regulon genes (pho1, pho84, tgp1). Whereas these STF alleles-asp1-386(Stop) and asp1-493(Stop)-were lethal in a wild-type CTD background, they were viable in combination with mutations in CPF and Rhn1, in which context Pho1 was also de-repressed. Our findings implicate Asp1 pyrophosphatase in constraining 1,5-IP8 or 1-IP7 synthesis by Asp1 kinase, without which 1-IPPs can accumulate to toxic levels that elicit precocious termination by CPF/Rhn1.
裂殖酵母磷酸盐稳态基因在富含磷酸盐的培养基中受到转录的长链非编码 RNA（lncRNA）的抑制，这些 lncRNA 干扰了侧翼 mRNA 启动子的激活。lncRNA 对 PHO 基因表达的调控受 RNA 聚合酶 II CTD 上的 Thr4 磷酸化位点和 3' 加工/终止因子 CPF 和 Rhn1 的影响，这些因子的突变导致 PHO 调控基因的过度抑制。在这里，我们进行了正向遗传筛选，以寻找解除 CTD-T4A 细胞中 Pho1 酸性磷酸酶表达抑制的突变。对 18 个独立的 STF（Thr4 四氨酸）分离株进行测序，结果表明，在每种情况下，Asp1 的 C 末端焦磷酸酶结构域都发生了突变，Asp1 是一种多功能的肌醇六磷酸（IPP）激酶/焦磷酸酶，可将 5-IPP 和 1,5-IPP8 相互转化。对两个 STF 菌株的重点特征分析确定了 51 个编码基因与 T4A 亲本菌株协调上调，包括所有三个 PHO 调控基因（pho1、pho84、tgp1）。虽然这些 STF 等位基因 asp1-386（终止）和 asp1-493（终止）在野生型 CTD 背景下是致死的，但它们在与 CPF 和 Rhn1 突变结合时是可行的，在这种情况下，Pho1 也被解除了抑制。我们的研究结果表明，Asp1 焦磷酸酶通过 Asp1 激酶限制 1,5-IPP8 或 1-IPP7 的合成，如果没有 Asp1 激酶，1-IPP 可以积累到毒性水平，从而引发 CPF/Rhn1 的过早终止。
