Genetic screen for suppression of transcriptional interference reveals fission yeast 14–3–3 protein Rad24 as an antagonist of precocious Pol2 transcription termination

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA-mediated interference is alleviated by genetic perturbations that elicit precocious lncRNA 3′-processing and transcription termination, such as (i) the inositol pyrophosphate pyrophosphatase-defective asp1-H397A allele, which results in elevated levels of IP8, and (ii) absence of the 14-3-3 protein Rad24. Combining rad24Δ with asp1-H397A causes a severe synthetic growth defect. A forward genetic screen for SRA (Suppressor of Rad24 Asp1-H397A) mutations identified a novel missense mutation (Tyr86Asp) of Pla1, the essential poly(A) polymerase subunit of the fission yeast cleavage and polyadenylation factor (CPF) complex. The pla1-Y86D allele was viable but slow-growing in an otherwise wild-type background. Tyr86 is a conserved active site constituent that contacts the RNA primer 3′ nt and the incoming ATP. The Y86D mutation elicits a severe catalytic defect in RNA-primed poly(A) synthesis in vitro and in binding to an RNA primer. Yet, analyses of specific mRNAs indicate that poly(A) tails in pla1-Y86D cells are not different in size than those in wild-type cells, suggesting that other RNA interactors within CPF compensate for the defects of isolated Pla1-Y86D. Transcriptome profiling of pla1-Y86D cells revealed the accumulation of multiple RNAs that are normally rapidly degraded by the nuclear exosome under the direction of the MTREC complex, with which Pla1 associates. We suggest that Pla1-Y86D is deficient in the hyperadenylation of MTREC targets that precedes their decay by the exosome.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

Inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8 are eukaryal signaling molecules that influence cell physiology, especially phosphate homeostasis. In fission yeast, 1,5-IP8 and 1-IP7 impact gene expression by acting as agonists of RNA 3'-processing and transcription termination. 1,5-IP8 is synthesized by position-specific kinases Kcs1 and Asp1 that convert IP6 to 5-IP7 and 5-IP7 to 1,5-IP8, respectively. Inositol pyrophosphatase enzymes Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase) are agents of inositol pyrophosphate catabolism in fission yeast. Whereas Asp1, Siw14, and Aps1 are individually inessential, double pyrophosphatase mutants asp1-H397A aps1∆ and siw14∆ aps1∆ display severe growth defects caused by overzealous 3'-processing/termination. By applying CE-ESI-MS to profile the inositol pyrophosphate content of fission yeast mutants in which inositol pyrophosphate toxicity is genetically suppressed, we elucidated the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Asp1, which exclusively cleaves the 1-β-phosphate, and Aps1, which prefers to cleave the 1-β-phosphate, play essential overlapping roles in guarding against the accumulation of toxic levels of 1-IP7. Aps1 and Siw14 together catabolize the inositol-5-pyrophosphates, and their simultaneous inactivation results in overaccumulation of 5-IP7. Cells lacking all three pyrophosphatases amass high levels of 1,5-IP8 and 1-IP7, with concomitant depletion of IP6. A genetic screen identified three missense mutations in the catalytic domain of Kcs1 kinase that suppressed inositol-1-pyrophosphate toxicosis. The screen also implicated the 3'-processing factor Swd22, the inositol pyrophosphate sensor Spx1, and the nuclear poly(A)-binding protein Nab2 as mediators of inositol-1-pyrophosphate toxicity.IMPORTANCEInositol pyrophosphates are key effectors of eukaryal cellular phosphate homeostasis. They are synthesized by kinases that add a β-phosphate to the 5- or 1-phosphate groups of IP6 and catabolized by three classes of pyrophosphatases that hydrolyze the β-phosphates of 5-IP7, 1-IP7, or 1,5-IP8. Whereas the fission yeast inositol pyrophosphatases-Asp1 (histidine acid phosphatase), Siw14 (cysteinyl phosphatase), and Aps1 (Nudix hydrolase)-are inessential for growth, Asp1/Aps1 and Aps1/Siw14 double mutations and Asp1/Siw14/Aps1 triple mutations elicit severe or lethal growth defects. By profiling the inositol pyrophosphate content of pyrophosphatase mutants in which this toxicity is genetically suppressed, we reveal the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Their synergies are manifested as excess accumulation of 1-IP7 upon dual inactivation of Asp1 and Aps1 or an excess of 5-IP7 in aps1∆ siw14∆ cells. In the absence of all three pyrophosphatases, cells accrue high levels of 1,5-IP8 and 1-IP7 while IP6 declines.

The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular 1,5-IP8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8. Genetic analyses implicate SpSiw14 in 1,5-IP8 catabolism in vivo, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14∆ aps1∆ lethality depends on synthesis of 1,5-IP8 by the Asp1 kinase. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of 3'-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP8 toxicosis.

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 selection 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 suppressor 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 ctf1Δ, swd22Δ, ppn1Δ, ssu72-C13S, rpb1-CTD-T4A, and asp1Δ establishes Msi2 as a central agent of 3'-processing/termination, functioning in parallel to inositol-1-pyrophosphates.

Tandem transcription interference occurs when the act of transcription from an upstream promoter suppresses utilization of a co-oriented downstream promoter. Because eukaryal genomes are liberally interspersed with transcription units specifying long non-coding (lnc) RNAs, there are many opportunities for lncRNA synthesis to negatively affect a neighboring protein-coding gene. Here, I review two eukaryal systems in which lncRNA interference with mRNA expression underlies a regulated biological response to nutrient availability. Budding yeast SER3 is repressed under serine-replete conditions by transcription of an upstream SRG1 lncRNA that traverses the SER3 promoter and elicits occlusive nucleosome rearrangements. SER3 is de-repressed by serine withdrawal, which leads to shut-off of SRG1 synthesis. The fission yeast phosphate homeostasis (PHO) regulon comprises three phosphate acquisition genes – pho1, pho84, and tgp1 – that are repressed under phosphate-replete conditions by 5′ flanking lncRNAs prt, prt2, and nc-tgp1, respectively. lncRNA transcription across the PHO mRNA promoters displaces activating transcription factor Pho7. PHO mRNAs are transcribed during phosphate starvation when lncRNA synthesis abates. The PHO regulon is de-repressed in phosphate-replete cells by genetic manipulations that favor ‘precocious’ lncRNA 3′-processing/termination upstream of the mRNA promoters. PHO lncRNA termination is governed by the Pol2 CTD code and is subject to metabolite control by inositol pyrophosphates.

Expression of fission yeast Pho1 acid phosphatase is repressed during growth in phosphate-rich medium. Repression is mediated by transcription of the prt locus upstream of pho1 to produce a long noncoding (lnc) prt RNA. Repression is also governed by RNA polymerase II CTD phosphorylation status, whereby inability to place a Ser7-PO4 mark (as in S7A) derepresses Pho1 expression, and inability to place a Thr4-PO4 mark (as in T4A) hyper-represses Pho1 in phosphate replete cells. Here we find that basal pho1 expression from the prt–pho1 locus is inversely correlated with the activity of the prt promoter, which resides in a 110-nucleotide DNA segment preceding the prt transcription start site. CTD mutations S7A and T4A had no effect on the activity of the prt promoter or the pho1 promoter, suggesting that S7A and T4A affect post-initiation events in prt lncRNA synthesis that make it less and more repressive of pho1, respectively. prt lncRNA contains clusters of DSR (determinant of selective removal) sequences recognized by the YTH-domain-containing protein Mmi1. Altering the nucleobase sequence of two DSR clusters in the prt lncRNA caused hyper-repression of pho1 in phosphate replete cells, concomitant with increased levels of the prt transcript. The isolated Mmi1 YTH domain binds to RNAs with single or tandem DSR elements, to the latter in a noncooperative fashion. We report the 1.75 Å crystal structure of the Mmi1 YTH domain and provide evidence that Mmi1 recognizes DSR RNA via a binding mode distinct from that of structurally homologous YTH proteins that recognize m6A-modified RNA.

Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24-Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence.

Saccharomyces cerevisiae adapts to oxidative, osmotic stress and nutrient deprivation through transcriptional changes, decreased proliferation, and entry into other developmental pathways such as pseudohyphal formation and sporulation. Inositol pyrophosphates are necessary for these cellular responses. Inositol pyrophosphates are molecules composed of the phosphorylated myo-inositol ring that carries one or more diphosphates. Mutations in the enzymes that metabolize these molecules lead to altered patterns of stress resistance, altered morphology, and defective sporulation. Mechanisms to alter the synthesis of inositol pyrophosphates have been recently described, including inhibition of enzyme activity by oxidation and by phosphorylation. Cells with increased levels of 5-diphosphoinositol pentakisphosphate have increased nuclear localization of Msn2 and Gln3. The altered localization of these factors is consistent with the partially induced environmental stress response and increased expression of genes under the control of Msn2/4 and Gln3. Other transcription factors may also exhibit increased nuclear localization based on increased expression of their target genes. These transcription factors are each regulated by TORC1, suggesting that TORC1 may be inhibited by inositol pyrophosphates. Inositol pyrophosphates affect stress responses in other fungi (Aspergillus nidulans, Ustilago maydis, Schizosaccharomyces pombe, and Cryptococcus neoformans), in human and mouse, and in plants, suggesting common mechanisms and possible novel drug development targets.

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.

ABSTRACTInositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1–385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1’s kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro, by virtue of its antagonism of IP8 turnover.

Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here, we show that phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3′-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of the Asp1 and Aps1 IPP pyrophosphatases is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1Δ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3′-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.

Noncoding RNAs (ncRNAs) are involved in various biological processes, including gene expression, development, and disease. Here, we identify a novel consensus sequence of a cis-element involved in long ncRNA (lncRNA) transcription and demonstrate that lncRNA transcription from this cis-element activates meiotic recombination via chromatin remodeling. In the fission yeast fbp1 gene, glucose starvation induces a series of promoter-associated lncRNAs, referred to as metabolic-stress-induced lncRNAs (mlonRNAs), which contribute to chromatin remodeling and fbp1 activation. Translocation of the cis-element required for mlonRNA into a well-characterized meiotic recombination hotspot, ade6-M26, further stimulates transcription and meiotic recombination via local chromatin remodeling. The consensus sequence of this cis-element (mlon-box) overlaps with meiotic recombination sites in the fission yeast genome. At one such site, the SPBC24C6.09c upstream region, meiotic double-strand break (DSB) formation is induced in an mlon-box-dependent manner. Therefore, mlonRNA transcription plays a universal role in chromatin remodeling and the regulation of transcription and recombination.

Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in β-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active β-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active β-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through β-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration.

The expression of the phosphate transporter Pho84 in fission yeast Schizosaccharomyces pombe is repressed in phosphate-rich medium and induced during phosphate starvation. Two other phosphate-responsive genes in S. pombe (pho1 and tgp1) had been shown to be repressed in cis by transcription of a long noncoding (lnc) RNA from the upstream flanking gene, but whether pho84 expression is regulated in this manner is unclear. Here, we show that repression of pho84 is enforced by transcription of the SPBC8E4.02c locus upstream of pho84 to produce a lncRNA that we name prt2 ( pho-repressive transcript 2). We identify two essential elements of the prt2 promoter, a HomolD box and a TATA box, mutations of which inactivate the prt2 promoter and de-repress the downstream pho84 promoter under phosphate-replete conditions. We find that prt2 promoter inactivation also elicits a cascade effect on the adjacent downstream prt (lncRNA) and pho1 (acid phosphatase) genes, whereby increased pho84 transcription down-regulates prt lncRNA transcription and thereby de-represses pho1 Our results establish a unified model for the repressive arm of fission yeast phosphate homeostasis, in which transcription of prt2, prt, and nc-tgp1 lncRNAs interferes with the promoters of the flanking pho84, pho1, and tgp1 genes, respectively.