Innokentev Aleksei, Sanchez Ana M, Bednor Lauren, Babor Jill, Schwer Beate, Shuman Stewart
Memorial Sloan Kettering Cancer Center.
Weill Cornell Medicine.
RNA. 2025 Sep 4. doi: 10.1261/rna.080664.125.
The fission yeast phosphate acquisition (PHO) regulon is repressed under phosphate-replete conditions by upstream lncRNA-mediated transcriptional interference. Inositol-1-pyrophosphates control phosphate homeostasis via their action as agonists of precocious PHO lncRNA 3'-processing/termination. Inositol pyrophosphatase-inactivating mutations that increase inositol-1-pyrophosphates elicit derepression of the PHO genes and a severe growth defect in YES medium. Previous studies demonstrated suppression of inositol pyrophosphate toxicosis by targeted deletion or loss-of-function mutations in the nonessential Ssu72, Ppn1, Swd22, and Ctf1 subunits of the fission yeast Cleavage and Polyadenylation Factor (CPF) complex. Here we conducted a screen for spontaneous mutations that suppress the precocious PHO lncRNA termination underlying the sickness of asp1-STF pyrophosphatase mutants. We thereby recovered and characterized novel hypomorphic missense mutations in five essential CPF subunits: Ysh1 (the cleavage endonuclease), Pta1 (an Armadillo/HEAT-repeat protein), Pfs2 (a WD repeat protein), Cft1 (a WD repeat protein), and Msi2 (a tandem RRM RNA-binding protein). The screen also yielded an intron branchpoint mutation in the gene encoding essential CPF subunit Iss1. In addition, we found that asp1-STF toxicosis was suppressed by a missense mutation in the active site of Pla1, the essential poly(A) polymerase subunit of CPF. Genetic crosses revealed a hierarchy of mutational synergies between the essential CPF subunits, the inessential CPF subunits, termination factor Rhn1, the Thr4 "letter" of the RNA polymerase II CTD code, and the Asp1 kinase that synthesizes inositol-1-pyrophosphates. The synthetic lethality of msi2-G252E with ctf1∆, swd22∆, ppn1∆, ssu72-C13S, rpb1-CTD-T4A, and asp1∆ establishes Msi2 as a central agent of 3'-processing/termination, functioning in parallel to inositol-1-pyrophosphates.
在磷酸盐充足的条件下，裂殖酵母的磷酸盐获取（PHO）调控子受到上游长链非编码RNA介导的转录干扰的抑制。肌醇-1-焦磷酸通过作为早熟PHO长链非编码RNA 3'加工/终止的激动剂来控制磷酸盐稳态。增加肌醇-1-焦磷酸的肌醇焦磷酸酶失活突变会导致PHO基因去抑制，并在YES培养基中产生严重的生长缺陷。先前的研究表明，通过靶向缺失或裂变酵母切割和聚腺苷酸化因子（CPF）复合物的非必需Ssu72、Ppn1、Swd22和Ctf1亚基的功能丧失突变，可以抑制肌醇焦磷酸中毒。在这里，我们对自发突变进行了筛选，这些突变可抑制asp1-STF焦磷酸酶突变体疾病背后的早熟PHO长链非编码RNA终止。我们由此在五个必需的CPF亚基中发现并鉴定了新的低功能错义突变：Ysh1（切割内切核酸酶）、Pta1（一种犰狳/热重复蛋白）、Pfs2（一种WD重复蛋白）、Cft1（一种WD重复蛋白）和Msi2（一种串联RRM RNA结合蛋白）。该筛选还在编码必需CPF亚基Iss1的基因中产生了一个内含子分支点突变。此外，我们发现CPF的必需聚腺苷酸聚合酶亚基Pla1活性位点的一个错义突变抑制了asp1-STF中毒。遗传杂交揭示了必需CPF亚基、非必需CPF亚基、终止因子Rhn1、RNA聚合酶II CTD密码的苏氨酸4“字母”以及合成肌醇-1-焦磷酸的Asp1激酶之间的突变协同层次。msi2-G252E与ctf1∆、swd22∆、ppn1∆、ssu72-C13S、rpb1-CTD-T4A和asp1∆的合成致死性确定Msi2是3'加工/终止的中心因子，与肌醇-1-焦磷酸并行发挥作用。
