Fission yeast poly(A) polymerase active site mutation Y86D alleviates therad24Δasp1-H397Asynthetic growth defect and up-regulates mRNAs targeted by MTREC and Mmi1

Abstract

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstreamprtlncRNA that interferes with thepho1mRNA 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-defectiveasp1-H397Aallele, which results in elevated levels of IP8, and (ii) absence of the 14-3-3 protein Rad24. Combiningrad24Δ withasp1-H397Acauses a severe synthetic growth defect. A forward genetic screen forSRA(Suppressor ofRad24Asp1-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. Thepla1-Y86Dallele 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 inpla1-Y86Dcells 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 ofpla1-Y86Dcells 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.