Fission yeast poly(A) polymerase active site mutation Y86D alleviates the  rad24 Δ  asp1-H397A  synthetic growth defect and up-regulates mRNAs targeted by MTREC and Mmi1.
Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream  prt  lncRNA that interferes with the  pho1  mRNA 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-defective  asp1-H397A  allele, which results in elevated levels of IP 8 , and (ii) absence of the 14-3-3 protein Rad24. Combining  rad24 Δ with  asp1-H397A  causes a severe synthetic growth defect. A forward genetic screen for  SRA  ( S uppressor of  R ad24  A sp1-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. The  pla1-Y86D  allele 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 in  pla1-Y86D  cells 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 of  pla1-Y86D  cells 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.
Garg A, Schwer B, Shuman S