Cleavage-Polyadenylation Factor Cft1 and SPX Domain Proteins Are Agents of Inositol Pyrophosphate Toxicosis in Fission Yeast

ABSTRACT Faithful chromosome segregation is fundamentally important for the maintenance of genome integrity and ploidy. By isolating conditional mutants defective in chromosome segregation in the fission yeast Schizosaccharomyces pombe, we identified a role for the essential gene pfs2 in chromosome dynamics. In the absence of functional Pfs2, chromosomal attachment to the mitotic spindle was defective, with consequent chromosome missegregation. Under these circumstances, multiple intracellular foci of spindle checkpoint proteins Bub1 and Mad2 were seen, and deletion of bub1 exacerbated the mitotic defects and the loss of cell viability that resulted from the loss of pfs2 function. Progression from G1 into S phase following release from nitrogen starvation also required pfs2 + function. The product of the orthologous Saccharomyces cerevisiae gene PFS2 is a component of a multiprotein complex required for 3′-end cleavage and polyadenylation of pre-mRNAs and, in keeping with the conservation of this essential function, an S. pombe pfs2 mutant was defective in mRNA 3′-end processing. Mutations in pfs2 were suppressed by overexpression of the putative mRNA 3′-end cleavage factor Cft1. These data suggest unexpected links between mRNA 3′-end processing and chromosome replication and segregation.

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A genetic screen for suppressors of hyper-repression of the fission yeast PHO regulon by Pol2 CTD mutation T4A implicates inositol 1-pyrophosphates as agonists of precocious lncRNA transcription termination

Nucleic Acids Research ◽

10.1093/nar/gkaa776 ◽

2020 ◽

Vol 48 (19) ◽

pp. 10739-10752

Author(s):

Angad Garg ◽

Stewart Shuman ◽

Beate Schwer

Keyword(s):

Fission Yeast ◽

Rna Polymerase Ii ◽

Genetic Screen ◽

Pho Regulon ◽

Wild Type ◽

Phosphate Homeostasis ◽

Inositol Pyrophosphate ◽

Inositol 1 ◽

Rna Polymerase Ii Ctd

Abstract 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.

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Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22

PLoS Genetics ◽

10.1371/journal.pgen.1009452 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009452

Author(s):

Bradley Benjamin ◽

Ana M. Sanchez ◽

Angad Garg ◽

Beate Schwer ◽

Stewart Shuman

Keyword(s):

Fission Yeast ◽

Rna Polymerase Ii ◽

Function Analysis ◽

Transcriptional Profiling ◽

Missense Mutations ◽

Rna Seq ◽

Phosphate Homeostasis ◽

Protein Coding ◽

Cleavage And Polyadenylation ◽

Polyadenylation Factor

Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3’ processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640–710. A TFIIS-like domain (aa 173–330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.

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Nitric oxide as a signaling molecule in the fission yeast Schizosaccharomyces pombe

PROTOPLASMA ◽

10.1007/s00709-009-0074-3 ◽

2009 ◽

Vol 238 (1-4) ◽

pp. 59-66 ◽

Cited By ~ 25

Author(s):

Cenk Kig ◽

Guler Temizkan

Keyword(s):

Nitric Oxide ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Signaling Molecule

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A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Journal of Visualized Experiments ◽

10.3791/59133 ◽

2019 ◽

Cited By ~ 1

Author(s):

Bahjat F. Marayati ◽

James B. Pease ◽

Ke Zhang

Keyword(s):

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Deep Sequencing ◽

Suppressor Screen

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Cleavage and polyadenylation factor, Rna14 is an essential protein required for the maintenance of genomic integrity in fission yeast Schizosaccharomyces pombe

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/j.bbamcr.2015.11.007 ◽

2016 ◽

Vol 1863 (2) ◽

pp. 189-197 ◽

Cited By ~ 3

Author(s):

Amit Sonkar ◽

Sudhanshu Yadav ◽

Shakil Ahmed

Keyword(s):

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Genomic Integrity ◽

Essential Protein ◽

Cleavage And Polyadenylation ◽

Polyadenylation Factor

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Genetic interactions and transcriptomics implicate fission yeast CTD prolyl isomerase Pin1 as an agent of RNA 3′ processing and transcription termination that functions via its effects on CTD phosphatase Ssu72

Nucleic Acids Research ◽

10.1093/nar/gkaa212 ◽

2020 ◽

Vol 48 (9) ◽

pp. 4811-4826 ◽

Cited By ~ 2

Author(s):

Ana M Sanchez ◽

Angad Garg ◽

Stewart Shuman ◽

Beate Schwer

Keyword(s):

Fission Yeast ◽

Transcriptional Profiling ◽

Prolyl Isomerase ◽

Protein Coding ◽

Peptidyl Prolyl Isomerase ◽

Growth Defects ◽

Repressive Effect ◽

Phosphorylation Pattern ◽

Ctd Phosphatase ◽

Polyadenylation Factor

Abstract The phosphorylation pattern of Pol2 CTD Y1S2P3T4S5P6S7 repeats comprises an informational code coordinating transcription and RNA processing. cis–trans isomerization of CTD prolines expands the scope of the code in ways that are not well understood. Here we address this issue via analysis of fission yeast peptidyl-prolyl isomerase Pin1. A pin1Δ allele that does not affect growth per se is lethal in the absence of cleavage-polyadenylation factor (CPF) subunits Ppn1 and Swd22 and elicits growth defects absent CPF subunits Ctf1 and Dis2 and termination factor Rhn1. Whereas CTD S2A, T4A, and S7A mutants thrive in combination with pin1Δ, a Y1F mutant does not, nor do CTD mutants in which half the Pro3 or Pro6 residues are replaced by alanine. Phosphate-acquisition genes pho1, pho84 and tgp1 are repressed by upstream lncRNAs and are sensitive to changes in lncRNA 3′ processing/termination. pin1Δ hyper-represses PHO gene expression and erases the de-repressive effect of CTD-S7A. Transcriptional profiling delineated sets of 56 and 22 protein-coding genes that are down-regulated and up-regulated in pin1Δ cells, respectively, 77% and 100% of which are downregulated/upregulated when the cis-proline-dependent Ssu72 CTD phosphatase is inactivated. Our results implicate Pin1 as a positive effector of 3′ processing/termination that acts via Ssu72.

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