Inositol pyrophosphates impact phosphate homeostasis via modulation of RNA 3′ processing and transcription termination

Abstract

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.

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

“…70% of the ppn1Δ/swd22Δ up-regulated mRNAs were coordinately up in dis2Δ cells; 43% of the ppn1Δ/swd22Δ down-regulated mRNAs were downregulated in dis2Δ cells. These transcriptomic data resonate with the DPS genetics, whereby ppn1Δ and swd22Δ display an identical spectrum of synthetic lethalities, which is broader than PLOS GENETICS that of dis2Δ [6,7]. All three DPS null mutants are synthetically lethal with the phosphatasedead alleles of CPF core subunit Ssu72 [6] and we see here that there was extensive overlap of the ppn1Δ/swd22Δ transcriptome with that of ssu72-C13S cells.…”

“…The basal Pho1 activity of wild-type cells was unaffected in ppn1-(173-710) cells [vis-à-vis the ppn1-(1-710) wild-type strain] but was hyper-repressed in ppn1-(1-496) cells expressing a non-functional Ppn1 mutant. The de-repression of Pho1 seen in CTD-S7A and asp1-H397A cells expressing wild-type Ppn1 was erased in the ppn1-(1-496) strain (Fig 10B), as shown previously for ppn1Δ [7]. A noteworthy finding was that the de-repressed levels of Pho1 activity in ppn1-(173-710) CTD-S7A and ppn1-(173-710) asp1-H397A cells were 38% https://doi.org/10.1371/journal.pgen.1009452.g010…”

“…Although DPS is inessential for vegetative growth, genetic analyses indicate that DPS deficiency is buffered by other factors that promote or affect 3’ processing and transcription termination, one of which is the CPF core subunit Ssu72 [ 6 ]. Recent RNA-seq studies show that inactivation of the Ssu72 protein phosphatase results in down-regulation or up-regulation of a narrow subset of fission yeast protein-coding genes [ 7 ]. By extension, we envision that: (i) the DPS complex also influences the expression of specific genes; and (ii) gauging the extent to which the effects of ablating individual DPS subunits on the transcriptome overlap and echo those of mutations in other transcription components might afford new insights to gene regulation.…”

“…This model is supported by findings that: (i) mutations of CPF subunits (including ppn1 Δ), Rhn1, and Pin1 –proteins that normally promote 3’ processing/termination–result in hyper-repression of pho1 under phosphate-replete conditions; and (ii) the de-repression of pho1 elicited by the CTD-S7A allele is erased by mutations of CPF subunits (including ppn1 Δ), Rhn1, and Pin1 [ 6 , 14 ]. Recent studies also implicate the inositol pyrophosphate (IPP) signaling molecule IP8 as an agent in the nexus of the CTD code with 3’ processing/termination, based on biochemical phenotypes and mutational synergies elicited by genetic manipulations of Asp1 [ 7 ], a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain [ 30 , 31 ]. The function of the Asp1 kinase is to generate 1,5-IP8 via phosphorylation of its substrate 5-IP7 and the function of the Asp1 pyrophosphatase is to convert its substrate 1,5-IP8 back to 5-IP7.…”

“…Moreover, Ssu72, Ctf1, Dis2, Swd22, and Ppn1 are constituents of a recently identified genetic interactome connecting 3’ processing/termination, the Pol2 “CTD code,” inositol pyrophosphate (IPP) signaling, and the transcriptional control of fission yeast phosphate homeostasis [ 6 , 7 ]. The carboxyl-terminal domain (CTD) of the Rpb1 subunit of fission yeast Pol2 consists of tandemly repeated heptapeptides of consensus sequence Y 1 S 2 P 3 T 4 S 5 P 6 S 7 .…”