RNA polymerase II CTD interactome with 3′ processing and termination factors in fission yeast and its impact on phosphate homeostasis

Abstract

The carboxy-terminal domain (CTD) code encrypted within the Y1S2P3T4S5P6S7heptad repeats of RNA polymerase II (Pol2) is deeply rooted in eukaryal biology. Key steps to deciphering the code are identifying the events in gene expression that are governed by individual “letters” and then defining a vocabulary of multiletter “words” and their meaning. Thr4 and Ser7 exert opposite effects on the fission yeastpho1gene, expression of which is repressed under phosphate-replete conditions by transcription of an upstream flanking long noncoding RNA (lncRNA). Here we attribute the derepression ofpho1by a CTD-S7Amutation to precocious termination of lncRNA synthesis, an effect that is erased by mutations of cleavage-polyadenylation factor (CPF) subunits Ctf1, Ssu72, Ppn1, Swd22, and Dis2 and termination factor Rhn1. By contrast, a CTD-T4Amutation hyperrepressespho1, as do CPF subunit and Rhn1 mutations, implying thatT4Areduces lncRNA termination. Moreover, CTD-T4Ais synthetically lethal withppn1∆ andswd22∆, signifying that Thr4 and the Ppn1•Swd22 module play important, functionally redundant roles in promoting Pol2 termination. We find that Ppn1 and Swd22 become essential for viability when the CTD array is curtailed and thatS7Aovercomes the need for Ppn1•Swd22 in the short CTD context. Mutational synergies highlight redundant essential functions of (i) Ppn1•Swd22 and Rhn1, (ii) Ppn1•Swd22 and Ctf1, and (iii) Ssu72 and Dis2 phosphatases. CTD allelesY1F,S2A, andT4Ahave overlapping synthetic lethalities withppn1∆ andswd22∆, suggesting that Tyr1-Ser2-Thr4 form a three-letter CTD word that abets termination, with Rhn1 being a likely “reader” of this word.

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

“…The asp1-D333A strain resembled asp1∆, except that it was able to form small colonies at 18˚C. Acid phosphatase activity (a gauge of Pho1 enzyme level that correlates with pho1 mRNA levels, as assayed by RT-qPCR, Northern blotting, primer extension, and RNA-seq [7,8,12,13,15]) was quantified by incubating suspensions of serial dilutions of the phosphate-replete cells for 5 min with p-nitrophenylphosphate and assaying colorimetrically the formation of p-nitrophenol. The basal Pho1 activity of wild-type rpb1-CTD cells was hyper-repressed by 4-fold in asp1∆ and asp1-D333A cells and de-repressed by 19-fold in asp1-H397A cells (Fig.…”

“…If this is the case, there are clear predictions concerning epistasis relationships between asp1-H397A and the 3' processing/termination machinery. Loss-of-function mutations in fission yeast proteins that promote cotranscriptional 3' processing and transcription termination hyperrepress Pho1 under phosphate-replete conditions (12). This hyper-repression is observed in knockout strains lacking the Dis2, Ctf1, Ppn1, or Swd22 subunits of the CPF complex, a strain with a catalytically dead (C13S) version of the Ssu72 protein phosphatase subunit of CPF, and a strain that lacks the transcription termination factor Rhn1 (12; and Fig.…”

“…The synthetic lethality accompanying simultaneous inactivation of any of seven pairwise combinations of CPF subunits and/or Rhn1 (these being ctf1∆ ppn1∆, ctf1∆ swd22∆, ppn1∆ rhn1∆, swd22∆ rhn1∆, ppn1∆ ssu72-C13S, swd22∆ ssu72-C13S, and dis2∆ ssu72-C13S; ref. 12) is posited to be the consequence of a severe termination defect impacting the expression of essential S. pombe genes. We asked whether such a lethal termination defect in a double-mutant might be ameliorated by a third mutation that exerts an opposite effect, e.g., the asp1-H397A allele that we hypothesize promotes precocious termination of the lncRNAs that control phosphate homeostasis.…”

“…Recent studies highlight 3' processing and transcription termination as a control point in the lncRNA-mediated repression of 3'-flanking gene expression. In particular, the prt-pho1 locus is established as a sensitive read-out of cellular influences on termination via their effect on the degree of interference with Pho1 expression by prt lncRNA transcription (12). Those influences include: the phosphorylation status of the RNA polymerase II (Pol2) carboxyl-terminal domain (CTD); the activity of protein kinases Csk1 and Cdk9; the Ctf1, Ssu72, Dis2, Ppn1, and Swd22 subunits of the CPF (cleavage polyadenylation factor) complex; and the transcription termination factor Rhn1 (3,7,8,12,13).…”

“…and (iv) the pho1 de-repressive CTD-S7A allele is synthetically lethal with the pho1 de-repressive cdk9 alleles T212A and T212E (12). We envision that there are additional influences on Pol2 transcription termination that are amenable to discovery via their impact on fission yeast phosphate homeostasis.…”