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

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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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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Development of a mariner-Based Transposon and Identification of Listeria monocytogenes Determinants, Including the Peptidyl-Prolyl Isomerase PrsA2, That Contribute to Its Hemolytic Phenotype

Journal of Bacteriology ◽

10.1128/jb.00016-09 ◽

2009 ◽

Vol 191 (12) ◽

pp. 3950-3964 ◽

Cited By ~ 70

Author(s):

Jason Zemansky ◽

Benjamin C. Kline ◽

Joshua J. Woodward ◽

Jess H. Leber ◽

Hélène Marquis ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Virulence Factor ◽

Ex Vivo ◽

Physiological Role ◽

Listeriolysin O ◽

Prolyl Isomerase ◽

Peptidyl Prolyl Isomerase ◽

Growth Defects ◽

Production Activity ◽

Cell Spread

ABSTRACT Listeriolysin O (LLO) is a pore-forming toxin that mediates phagosomal escape and cell-to-cell spread of the intracellular pathogen Listeria monocytogenes. In order to identify factors that control the production, activity, or secretion of this essential virulence factor, we constructed a Himar1 mariner transposon delivery system and screened 50,000 mutants for a hypohemolytic phenotype on blood agar plates. Approximately 200 hypohemolytic mutants were identified, and the 51 most prominent mutants were screened ex vivo for intracellular growth defects. Eight mutants with a phenotype were identified, and they contained insertions in the following genes: lmo0964 (similar to yjbH), lmo1268 (clpX), lmo1401 (similar to ymdB), lmo1575 (similar to ytqI), lmo1695 (mprF), lmo1821 (similar to prpC), lmo2219 (prsA2), and lmo2460 (similar to cggR). Some of these genes are involved in previously unexplored areas of research with L. monocytogenes: the genes yjbH and clpX regulate the disulfide stress response in Bacillus subtilis, and the prpC phosphatase has been implicated in virulence in other gram-positive pathogens. Here we demonstrate that prsA2, an extracytoplasmic peptidyl-prolyl cis/trans isomerase, is critical for virulence and contributes to the folding of LLO and to the activity of another virulence factor, the broad-range phospholipase C (PC-PLC). Furthermore, although it has been shown that prsA2 expression is linked to PrfA, the master virulence transcription factor in L. monocytogenes pathogenesis, we demonstrate that prsA2 is not directly controlled by PrfA. Finally, we show that PrsA2 is involved in flagellum-based motility, indicating that this factor likely serves a broad physiological role.

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Uncovering Natural Longevity Alleles from Intercrossed Pools of Aging Fission Yeast Cells

10.1101/352583 ◽

2018 ◽

Author(s):

David A. Ellis ◽

Ville Mustonen ◽

María Rodríguez-López ◽

Charalampos Rallis ◽

Michał Malecki ◽

...

Keyword(s):

Fission Yeast ◽

Genetic Variants ◽

Genetic Factors ◽

Cellular Aging ◽

Yeast Cells ◽

Untranslated Regions ◽

Protein Coding ◽

Chronological Lifespan ◽

Growth Defects ◽

Chromosome Ii

ABSTRACTQuantitative traits often show large variation caused by multiple genetic factors. One such trait is the chronological lifespan of non-dividing yeast cells, serving as a model for cellular aging. Screens for genetic factors involved in ageing typically assay mutants of protein-coding genes. To identify natural genetic variants contributing to cellular aging, we exploited two strains of the fission yeast, Schizosaccharomyces pombe, that differ in chronological lifespan. We generated segregant pools from these strains and subjected them to advanced intercrossing over multiple generations to break up linkage groups. We chronologically aged the intercrossed segregant pool, followed by genome sequencing at different times to detect genetic variants that became reproducibly enriched as a function of age. A region on Chromosome II showed strong positive selection during ageing. Based on expected functions, two candidate variants from this region in the long-lived strain were most promising to be causal: small insertions and deletions in the 5’-untranslated regions of ppk31 and SPBC409.08. Ppk31 is an orthologue of Rim15, a conserved kinase controlling cell proliferation in response to nutrients, while SPBC409.08 is a predicted spermine transmembrane transporter. Both Rim15 and the spermine-precursor, spermidine, are implicated in ageing as they are involved in autophagy-dependent lifespan extension. Single and double allele replacement suggests that both variants, alone or combined, have subtle effects on cellular longevity. Furthermore, deletion mutants of both ppk31 and SPBC409.08 rescued growth defects caused by spermidine. We propose that Ppk31 and SPBC409.08 may function together to modulate lifespan, thus linking Rim15/Ppk31 with spermidine metabolism.

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