Inositol pyrophosphate dynamics affect RNA 3'-processing/transcription termination

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ABSTRACT Inositol pyrophosphates (IPPs) are signaling molecules involved in diverse cellular processes from telomere maintenance and apoptosis to vesicular trafficking and cell migration. Alterations in IPP levels (via mutations in IPP metabolizing enzymes) are linked to human pathology including cancer, obesity, diabetes and hearing loss. The pleiotropic effects suggest that inositol pyrophosphates have the ability to control very basic cellular functions. A role for IPPs in phosphate sensing and phosphate homeostasis is documented in fungi, plants, and humans. Cells respond to phosphate limitation by inducing the transcription of phosphate acquisition genes, yet the mechanisms by which this is achieved differ in each taxon. The phosphate (PHO) regulon in the fission yeast Schizosaccharomyces pombe comprises three genes that specify, respectively, a cell surface acid phosphatase Pho1, an inorganic phosphate transporter Pho84, and a glycerophosphate transporter Tgp1. Expression of pho1, pho84, and tgp1 is actively repressed during growth in phosphate-rich medium by RNA polymerase II (Pol2) transcription in cis of a long noncoding (lnc) RNA from the respective 5' flanking genes prt, prt2, and nc-tgp1. lncRNA transcription across the PHO mRNA promoter displaces the activating transcription factor Pho7 and thereby interferes with PHO mRNA expression. The system of lncRNA-mediated transcriptional interference is sensitive to genetic manipulations that influence 3’-processing/termination. Mutations that elicit “precocious” lncRNA 3'-processing/termination in response to poly(A) signals upstream of the mRNA promoters lead to de- repression of pho1, whereas genetic changes that diminish lncRNA termination hyper-repress pho1 expression. We have exploited this system to discover novel influences on 3’-processing and Pol2 termination. We showed that: (i) lncRNA transcription is subject to metabolite control by inositol pyrophosphate 1,5-IP8, which acts as an agonist of precocious 3'-processing/termination; (ii) Spx1, which is composed of an SPX domain and a RING E3 ligase domain, is a likely mediator of IP8 signaling to the 3'-processing/termination machinery; (iii) the 14-3-3 protein Rad24 antagonizes precocious 3’-processing/termination in a manner that depends on its phosphate binding site; and (iv) Pol2 termination can be enhanced via a gain-of-function mutation in the essential termination factor Seb1. Specific aims are: (1) to identify targets of the Spx1 E3 ubiquitin ligase (via TULIP2 tagging and mass spectrometry); and to test whether pyrophosphorylation of (or IP8 binding to) components of the Pol2 machinery contributes to the effects of IP8 on 3’-processing/termination; (2) to leverage new analytical methods to assess IPP abundance and isoform distribution in fission yeast strains where genetics predicts changes in IPP content, and to investigate whether and how phosphate starvation impacts cellular IPP content; (3) To probe the mechanism by which Rad24 antagonizes 3’-processing/termination, by performing ChIP-seq to identify genes with which Rad24 is associated in vivo; and (4) To generate new gain-of-function mutations in the essential termination factor Seb1 and analyze their effects on poly(A) site usage, RNA binding, CTD binding.