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. IPPs are known to participate in phosphate sensing and phosphate homeostasis in yeast, plant and mammalian cells. Fungi respond to phosphate starvation by inducing the transcription of phosphate acquisition genes. The phosphate 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 the transcription in cis of a long noncoding (lnc) RNA from the respective 5' flanking genes prt, prt2, and nc-tgp1. It is proposed that transcription of the upstream lncRNA interferes with expression of the downstream mRNA genes by displacing the activating transcription factor Pho7 from its binding site(s) in the mRNA promoters. The key discoveries underlying the present proposal are our findings that: (i) 3?-processing and transcription termination is a control point in the lncRNA- mediated repression of 3?-flanking gene expression, and (ii) Pho1 expression from the prt?pho1 locus is a sensitive read-out of cellular influences on termination. Based on these findings, we hypothesize that IPP dynamics affect 3?-processing/transcription termination and influence poly(A) site usage.
Specific aims are to: (1) use genetic array analyses and reveal the extent to which the functions of 3? processing/transcription termination factors, the RNA Pol II CTD, and factors involved in sculpting the CTD phosphorylation array depend on IPP levels; (2) assess ? at the genome-wide level ? the impact of IPP dynamics on gene expression and 3?-end formation, by analyzing mRNA and nascent RNA profiles and mapping poly(A) sites in wild-type cells and in cells with altered IPP levels; and (3) explore mechanisms by which IPPs influence Pol2 transcription termination. Using in vitro synthesized IPPs, we will test whether components of the 3?-processing/transcription termination machinery are targets for pyrophosphorylation and whether IPPs affect the activities of CTD kinases. We expect to gain new and general insights into the role of these important signaling molecules in gene expression, and to illuminate the signal transduction pathway involved in fission yeast phosphate homeostasis.
? One of the fundamental and highly regulated events in the generation of functional mRNAs is 3?-processing and transcription termination. The proposal explores the role of small metabolites (inositol pyrophosphates) in mRNA 3?-end formation through genetic and biochemical studies in an effort to illuminate molecular mechanisms that underlie defects linked to human diseases including cancer.
